Pathway analysis computing system and method

ABSTRACT

The present invention relates to methods for evaluating the probability that a patient&#39;s diagnosis may be treated with a particular clinical regimen or therapy.

RELATIONSHIP TO OTHER APPLICATIONS

This application is related to and claims priority to U.S.Non-Provisional patent application Ser. No. 13/068,002 entitled “PATHWAYRECOGNITION ALGORITHM USING DATA INTEGRATION ON GENOMIC MODELS(PARADIGM)” filed Apr. 29, 2011, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/343,575 entitled “PATHWAYRECOGNITION ALGORITHM USING DATA INTEGRATION ON GENOMIC MODELS(PARADIGM)” filed 29 Apr. 2010, which is herein incorporated byreference in its entirety.

This invention was made partly using funds from the following UnitedStated Federal agencies: NSF CAREER award 0845783, National CancerInstitute Contract/Grant numbers 5R21CA135937-02 and 1U24CA143858-01,and National Institute of Health Training Grant number T32 GM070386-01.The US Federal Government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to a method for identifying components ofbiological pathways in an individual or subject and determining if theindividual or subject is a candidate for a clinical regimen ortreatment. The invention also relates to using the methods to diagnosewhether a subject is susceptible to cancer, autoimmune diseases, cellcycle disorders, or other disorders.

BACKGROUND

A central premise in modern cancer treatment is that patient diagnosis,prognosis, risk assessment, and treatment response prediction can beimproved by stratification of cancers based on genomic, transcriptionaland epigenomic characteristics of the tumor alongside relevant clinicalinformation gathered at the time of diagnosis (for example, patienthistory, tumor histology and stage) as well as subsequent clinicalfollow-up data (for example, treatment regimens and disease recurrenceevents).

While several high-throughput technologies have been available forprobing the molecular details of cancer, only a handful of successeshave been achieved based on this paradigm. For example, 25% of breastcancer patients presenting with a particular amplification oroverexpression of the ERBB2 growth factor receptor tyrosine kinase cannow be treated with trastuzumab, a monoclonal antibody targeting thereceptor (Vogel C, Cobleigh M A, Tripathy D, Gutheil J C, Harris L N,Fehrenbacher L, Slamon D J, Murphy M, Novotny W F, Burchmore M, Shak S,Stewart S J. First-line, single-agent Herceptin® (trastuzumab) inmetastatic breast cancer. A preliminary report. Eur. J. Cancer 2001January; 37 Suppl 1:2529).

However, even this success story is clouded by the fact that fewer than50% of patients with ERBB2-positive breast cancers actually achieve anytherapeutic benefit from trastuzumab, emphasizing our incompleteunderstanding of this well-studied oncogenic pathway and the manytherapeutic-resistant mechanisms intrinsic to ERBB2-positive breastcancers (Park J W, Neve R M, Szollosi J, Benz C C. Unraveling thebiologic and clinical complexities of HER2. Clin. Breast Cancer 2008October; 8(5):392-401.)

This overall failure to translate modern advances in basic cancerbiology is in part due to our inability to comprehensively organize andintegrate all of the omic features now technically acquirable onvirtually any type of cancer. Despite overwhelming evidence thathistologically similar cancers are in reality a composite of manymolecular subtypes, each with significantly different clinical behavior,this knowledge is rarely applied in practice due to the lack of robustsignatures that correlate well with prognosis and treatment options.

Cancer is a disease of the genome that is associated with aberrantalterations that lead to disregulation of the cellular system. What isnot clear is how genomic changes feed into genetic pathways thatunderlie cancer phenotypes. High-throughput functional genomicsinvestigations have made tremendous progress in the past decade(Alizadeh A A, Eisen M B, Davis R E, Ma C, Lossos I S, Rosenwald A,Boldrick J C, Sabet H, Tran T, Yu X, Powell J I, Yang L, Marti G E,Moore T, Hudson J, Lu L, Lewis D B, Tibshirani K SHERLOCK G, Chan W C,Greiner T C, Weisenburger D D, Armitage J O, Wamke R, Levy R, Wilson W,Greyer M R, Byrd J C, Botstcin D, Brown P O, Staudt L M. Distinct typesof diffuse large B-cell lymphoma identified by gene expressionprofiling. Nature 2000 February; 403(6769):503-511.; Golub T R, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov J P, Coller H, Loh M L,Downing J R, Caligiuri M A, Bloomfield C D, Lander E S. Molecularclassification of cancer: class discovery and class prediction by geneexpression monitoring. Science 1999 October; 286(5439):531-537.; van dcVijver M J, He Y D, van t Veer L I, Dai H. Hart A A M, Voskuil D W,Schreiber G J, Peterse J L, Roberts C, Marton M J, Parrish M, Atsma D,Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, RodenhuisS, Rutgers E T, Friend S H, Bernards R. A Gene-Expression Signature as aPredictor of Survival in Breast Cancer. N Engl J Med 2002December:347(25):1999-2009.)

However, the challenges of integrating multiple data sources to identifyreproducible and interpretable molecular signatures of tumorigenesis andprogression remain elusive. Recent pilot studies by TCGA and others makeit clear that a pathway-level understanding of genomic perturbations isneeded to understand the changes observed in cancer cells. Thesefindings demonstrate that even when patients harbor genomic alterationsor aberrant expression in different genes, these genes often participatein a common pathway. In addition, and even more striking, is that thealterations observed (for example, deletions versus amplifications)often alter the pathway output in the same direction, either allincreasing or all decreasing the pathway activation. (See Parsons D W,Jones S, Zhang X, Lin J C H, Leary R J, Angenendt P, Mankoo P, Carter H,Siu I, Gallia G L, Olivi A, McLendon R, Rasheed B A, Keir S, NikolskayaT, Nikolsky Y, Busam D A, Tekleab H, Diaz L A, Hartigan J, Smith D R,Strausberg R L, Marie S K N, Shinjo S M O, Yan H, Riggins G J, Bigner DD, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler K W. An Integrated Genomic Analysis of Human GlioblastomaMultiforme. Science 2008 September; 321(5897):1807-1812.; Cancer GenomeAtlas Research Network. Comprehensive genomic characterization defineshuman glioblastoma genes and core pathways. Nature 2008 October;455(7216):1061-1068.)

Approaches for interpreting genome-wide cancer data have focused onidentifying gene expression profiles that are highly correlated with aparticular phenotype or disease state, and have led to promisingresults. Methods using analysis of variance, false-discovery, andnon-parametric methods have been proposed. (See Troyanskaya et al.,2002) have been proposed. Allison D B, Cui X, Page G P, Sabripour M.Microarray data analysis: from disarray to consolidation and consensus.Nat. Rev. Genet. 2006 January; 7(1):55-65.; Dudoit S, Fridlyand J. Aprediction-based resampling method for estimating the number of clustersin a dataset. Genome Biol 2002 June; 3(7):RESEARCH0036-RESEARCH0036.21.;Tusher V G, Tibshirani R, Chu G. Significance analysis of microarraysapplied to the ionizing radiation response. Proc. Natl. Acad. Sci.U.S.A. 2001 April; 98(9):5116-5121; Kerr M K, Martin M, Churchill G A.Analysis of variance for gene expression microarray data. J. Comput.Biol. 2000; 7(6):819-837; Storey J D, Tibshirani R. Statisticalsignificance for genomewide studies. Proc. Natl. Acad. Sci. U.S.A. 2003August; 100(16):9440-9445; and Troyanskaya O G, Garber M E, Brown P O,Botstein D, Altman R B. Nonparametric methods for identifyingdifferentially expressed genes in microarray data. Bioinformatics 2002November; 18(11):1454-1461.)

Several pathway-level approaches use statistical tests based onoverrepresentation of genesets to detect whether a pathway is perturbedin a disease condition. In these approaches, genes are ranked based ontheir degree of differential activity, for example as detected by eitherdifferential expression or copy number alteration. A probability scoreis then assigned reflecting the degree to which a pathway's genes ranknear the extreme ends of the sorted list, such as is used in gene setenrichment analysis (GSEA) (Subramanian A, Tamayo P, Mootha V K,Mukherjee S, Ebert B L, Gillette M A, Paulovich A, Pomeroy S L, Golub TR, Lander E S, Mesirov J P. Gene set enrichment analysis: aknowledge-based approach for interpreting genome-wide expressionprofiles. Proc. Natl. Acad. Sci. U.S.A. 2005 October;102(43):15545-15550.). Other approaches include using a hypergeometrictest-based method to identify Gene Ontology (Ashbumer M, Ball C A, BlakeJ A, Botstein D, Butler H, Cherry J M, Davis A P, Dolinski K, Dwight SS, Eppig J T, Harris M A, Hill D P, Issel-Tarver L, Kasarskis A, LewisS, Matese J C, Richardson J E, Ringwald M, Rubin G M, SHERLOCK G. Geneontology: tool for the unification of biology. The Gene OntologyConsortium. Nat Genet 2000 May; 25(1):25-29.) or MIPS mammalianprotein-protein interaction (Pagel P, Kovac S, Oesterheld M, Brauner B,Dunger-Kaltenbach I, Frishman G, Montrone C, Mark P, Sttimpflen V, MewesH, Ruepp A, Frishman D. The MIPS mammalian protein-protein interactiondatabase. Bioinformatics 2005 March; 21(6):832-834.) categories enrichedin differentially expressed genes (Tamayo P, Slonim D, Mesirov J, Zhu Q,Kitareewan S, Dmitrovsky E, Lander E S, Golub T R. Interpreting patternsof gene expression with self-organizing maps: methods and application tohematopoietic differentiation. Proc. Natl. Acad. Sci. U.S.A. 1999 March;96(6):2907-2912.).

Overrepresentation analyses are limited in their efficacy because theydo not incorporate known interdependencies among genes in a pathway thatcan increase the detection signal for pathway relevance. In addition,they treat all gene alterations as equal, which is not expected to bevalid for many biological systems.

Further complicating the issue is the fact that many genes (for example,microRNAs) are pleiotropic, acting in several pathways with differentroles (Maddika S, Ande S R, Panigrahi S, Paranjothy T, Weglarczvk K,Zuse A, Eshraghi M, Manda K D, Wiechec E, Los M. Cell survival, celldeath and cell cycle pathways are interconnected: implications forcancer therapy. Drug Resist. Updat. 2007 January; 10(1-2):13-29).Because of these factors, overrepresentation analyses often missfunctionally-relevant pathways whose genes have borderline differentialactivity. They can also produce many false positives when only a singlegene is highly altered in a small pathway. Our collective knowledgeabout the detailed interactions between genes and their phenotypicconsequences is growing rapidly.

While the knowledge was traditionally scattered throughout theliterature and hard to access systematically, new efforts are catalogingpathway knowledge into publicly available databases. Some of thedatabases that include pathway topology are Reactome (Joshi-Tope G,Gillespie M, Vastrik 1, D'Eustachio P, Schmidt E, de Bono B, Jassal B,Gopinath G R, Wu G R. Matthews L, Lewis S, Birney E, Stein L. Reactome:a knowledgebase of biological pathways. Nucleic Acids Res. 2005 January;33(Database issue):D428-32; Ogata H, Goto S, Sato K, Fujibuchi W, BonoH, Kanehisa M. KEGG: Kyoto Encyclopedia of Genes and Genomes. NucleicAcids Res. 1999 January; 27(1):29-34.)) and the NCI Pathway InteractionDatabase. Updates to these databases are expected to improve ourunderstanding of biological systems by explicitly encoding how genesregulate and communicate with one another. A key hypothesis is that theinteraction topology of these pathways can be exploited for the purposeof interpreting high-throughput datasets.

Until recently, few computational approaches were available forincorporating pathway knowledge to interpret high-throughput datasets.However, several newer approaches have been proposed that incorporatepathway topology (Efroni S, Schaefer C F, Buetow K H. Identification ofkey processes underlying cancer phenotypes using biologic pathwayanalysis. PLoS ONE 2007; 2(5):e425.). One approach, called SignalingPathway Impact Analysis (SPIA), uses a method analogous to Google'sPageRank to determine the influence of a gene in a pathway (Tarca A L,Draghici S, Khatri P, Hassan S S, Mittal P, Kim J, Kim C J, Kusanovic JP, Romero R. A novel signaling pathway impact analysis. Bioinformatics2009 January; 25(1):75-82.) In SPIA, more influence is placed on genesthat link out to many other genes. SPIA was successfully applied todifferent cancer datasets (lung adenocarcinoma and breast cancer) andshown to outperform overrepresentation analysis and Gene Set EnrichmentAnalysis for identifying pathways known to be involved in these cancers.While SPIA represents a major step forward in interpreting cancerdatasets using pathway topology, it is limited to using only a singletype of gcnome-wide data.

New computational approaches are needed to connect multiple genomicalterations such as copy number, DNA methylation, somatic mutations,mRNA expression and microRNA expression. Integrated pathway analysis isexpected to increase the precision and sensitivity of causalinterpretations for large sets of observations since no single datasource is likely to provide a complete picture on its own.

In the past several years, approaches in probabilistic graphical models(PGMs) have been developed for learning causal networks compatible withmultiple levels of observations. Efficient algorithms are available tolearn pathways automatically from data (Friedman N, Goldszmidt M. (1997)Sequential Update of Bayesian Network Structure. In: Proceedings of theThirteenth Conference on Uncertainty in Artificial Intelligence(UAI'97), Morgan Kaufinann Publishers, pp. 165-174; Murphy K, Weiss Y.Loopy belief propagation for approximate inference: An empirical study.In: Proceedings of Uncertainty in AI. 1999) and are well adapted toproblems in genetic network inference (Friedman N. Inferring cellularnetworks using probabilistic graphical models. Science 2004 February;303(5659):799-805.). As an example, graphical models have been used toidentify sets of genes that form ‘modules’ in cancer biology (Segal E,Friedman N, Kaminski N, Regev A, Koller D. From signatures to models:understanding cancer using microarrays. Nat Genet 2005 June; 37Suppl:S38-45.). They have also been applied to elucidate therelationship between tumor genotype and expression phenotypes (Lee S,Pe'er D, Dudley A M, Church G M, Koller D. Identifying regulatorymechanisms using individual variation reveals key role for chromatinmodification. Proc. Natl. Acad. Sci. U.S.A. 2006 September;103(38):14062-14067.), and infer protein signal networks (Sachs K, PerezO, Pe'er D, Lauffenburger D A, Nolan G P. Causal protein-signalingnetworks derived from multiparameter single-cell data. Science 2005April; 308(5721):523-529.) and recombinatorial gene regulatory code(Beer M A, Tavazoie S. Predicting gene expression from sequence. Cell2004 April; 117(2):185-198.). In particular, factor graphs have beenused to model expression data (Gat-Viks I, Shamir R. Refinement andexpansion of signaling pathways: the osmotic response network in yeast.Genome Research 2007 March; 17(3):358-367.; Gat-Viks I, Tanay A, RaijmanD, Shamir R. The Factor Graph Network Model for Biological Systems. In:Hutchison D, Kanade T, Kittler J, Kleinberg J M, Mattem F, Mitchell J C,Naor M, Nierstrasz O, Pandu Rangan C, Steffen B, Sudan M, Terzopoulos D,Tygar D, Vardi M Y, Weikum G, Mivano S. Mesirov J, Kasif S, Istrail S,Pevzner P A, Waterman M, editors. Berlin, Heidelberg: Springer BerlinHeidelberg; 2005 p. 31-47.; Gat-Viks I, Tanay A, Raijman D, Shamir R. Aprobabilistic methodology for integrating knowledge and experiments onbiological networks. J. Comput. Biol. 2006 March; 13(2):165-181.).

Breast cancer is clinically and genomically heterogeneous and iscomposed of several pathologically and molecularly distinct subtypes.Patient responses to conventional and targeted therapeutics differ amongsubtypes motivating the development of marker guided therapeuticstrategies. Collections of breast cancer cell lines mirror many of themolecular subtypes and pathways found in tumors, suggesting thattreatment of cell lines with candidate therapeutic compounds can guideidentification of associations between molecular subtypes, pathways anddrug response. In a test of 77 therapeutic compounds, nearly all drugsshow differential responses across these cell lines and approximatelyhalf show subtype-, pathway and/or genomic aberration-specificresponses. These observations suggest mechanisms of response andresistance that may inform clinical drug deployment as well as effortsto combine drugs effectively.

The accumulation of high throughput molecular profiles of tumors atvarious levels has been a long and costly process worldwide. Combinedanalysis of gene regulation at various levels may point to specificbiological functions and molecular pathways that are deregulated inmultiple epithelial cancers and reveal novel subgroups of patients fortailored therapy and monitoring. We have collected high throughput dataat several molecular levels derived from fresh frozen samples fromprimary tumors, matched blood, and with known micrometastases status,from approximately 110 breast cancer patients (further referred to asthe MicMa dataset). These patients are part of a cohort of over 900breast cancer cases with information about presence of disseminatedtumor cells (DTC), long-term follow-up for recurrence and overallsurvival. The MicMa set has been used in parallel pilot studies of wholegenome mRNA expression (I Naume, B. et al., (2007), Presence of bonemarrow micrometastasis is associated with different recurrence riskwithin molecular subtypes of breast cancer, 1: 160-171), arrayCGH(Russnes H G, Vollan H K M, Lingjaerde O C, Krasnitz A, Lundin P, NaumeB, lie T, Borgen E, Rye I H, LangerOcl A, Chin S, Teschendorff A E,Stephens P J, MAner S, Schlichting E, Baumbusch L O, KAresen R, StrattonM P, Wigler M, Caldas C, Zetterberg A, Hicks J, Borresen-Dale A. Genomicarchitecture characterizes tumor progression paths and fate in breastcancer patients. Sci Transl Med 2010 June; 2(38):38ra47), DNAmethylation (Ronneberg J A. Fleischer T, Solvang H K, Nordgard S H,Edvardsen H, Potapenko I, Nebdal D, Daviaud C, Gut I, Bukholm I, NaumeB, Borresen-Dale A, Tost J, Kristensen V. Methylation profiling with apanel of cancer related genes: association with estrogen receptor, TP53mutation status and expression subtypes in sporadic breast cancer. MolOncol 2011 February; 5(1):61-76), whole genome SNP and SNP-CGH (Van, LooP. et al., (2010), Allele-specific copy number analysis of tumors, 107:16910-169154), whole genome miRNA expression analyses (5 Enerly, E. etal., (2011), miRNA-mRNA Integrated Analysis Reveals Roles for miRNAs inPrimary Breast Tumors, 6: e 16915-), TP53 mutation status dependentpathways and high throughput paired end sequencing (7 Stephens, P. J. etal., (2009), Complex landscapes of somatic rearrangement in human breastcancer genomes, 462: 1005-1010). This is a comprehensive collection ofhigh throughput molecular data performed by a single lab on the same setof primary tumors of the breast.

A topic of great importance in cancer research is the identification ofgenomic aberrations that drive the development of cancer. Utilizingwhole-genome copy number and expression profiles from the MicMa cohort,we defined several filtering steps, each designed to identify the mostpromising candidates among the genes selected in the previous step. Thefirst two steps involve identification of commonly aberrant and in-ciscorrelated to expression genes, i.e. genes for which copy number changeshave substantial effect on expression. Subsequently, the methodconsiders in-trans effects of the selected genes to further narrow downthe potential novel candidate driver genes (Miriam Ragle Aure, IsraelSteinfeld Lars Oliver Baumbusch Knut Liestol Doron Lipson Bjorn NaumeVessela N. Kristensen Anne-Lise Borresen-Dale Ole-Christian Lingjwrdeand Zohar Yakhini, (2011), A robust novel method for the integratedanalysis of copy number and expression reveals new candidate drivergenes in breast cancer). Recently we developed an allele-specific copynumber analysis enabling us to accurately dissect the allele-specificcopy number of solid tumors (ASCAT), and simultaneously estimating andadjusting for both tumor ploidy and nonaberrant cell admixture (Van, LooP. et al., (2010), Allele-specific copy number analysis of tumors, 107:16910-169154). This allows calculation of genome-wide allele-specificcopy-number profiles from which gains, losses, copy number-neutralevents, and loss of heterozygosity (LOH) can accurately be determined.Observing DNA aberrations in allele specific manner allowed us toconstruct a genome-wide map of allelic skewness in breast cancer,indicating loci where one allele is preferentially lost, whereas theother allele is preferentially gained. We hypothesize that thesealternative alleles have a different influence on breast carcinomadevelopment. We could also see that Basal-like breast carcinomas have asignificantly higher frequency of LOH compared with other subtypes, andtheir ASCAT profiles show large-scale loss of gcnomic material duringtumor development, followed by a whole-genome duplication, resulting innear-triploid genomes (Van et al. (2010) supra). Distinct global DNAmethylation profiles have been reported in normal breast epithelialcells as well as in breast tumors.

There is currently a need to provide methods that can be used incharacterization, diagnosis, prevention, treatment, and determiningoutcome of diseases and disorders.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a method of generating adynamic pathway map (DPM), the method comprising: providing access to apathway element database storing a plurality of pathway elements, eachpathway element being characterized by its involvement in at least onepathway; providing access to a modification engine coupled to thepathway element database; using the modification engine to associate afirst pathway element with at least one a priori known attribute; usingthe modification engine to associate a second pathway element with atleast one assumed attribute; using the modification engine tocross-correlate and assign an influence level of the first and secondpathway elements for at least one pathway using the known and assumedattributes, respectively, to form a probabilistic pathway model; andusing the probabilistic pathway model, via an analysis engine, to derivefrom a plurality of measured attributes for a plurality of elements of apatient sample the DPM having reference pathway activity information fora particular pathway. In one preferred embodiment, the pathway elementis a protein. In a more preferred embodiment, the protein is selectedfrom the group consisting of a receptor, a hormone binding protein, akinase, a transcription factor, a methylase, a histone acetylase, and ahistone deacetylase. In an alternative preferred embodiment, the pathwayelement is a nucleic acid. In a more preferred embodiment, the nucleicacid is selected from the group consisting of a protein coding sequence,a genomic regulatory sequence, a regulatory RNA, and a trans-activatingsequence. In another more preferred embodiment, the reference pathwayactivity information is specific with respect to a normal tissue, adiseased tissue, an ageing tissue, or a recovering tissue. In apreferred embodiment, the known attribute is selected from the groupconsisting of a compound attribute, a class attribute, a gene copynumber, a transcription level, a translation level, and a proteinactivity. In another preferred embodiment, the assumed attribute isselected from the group consisting of a compound attribute, a classattribute, a gene copy number, a transcription level, a translationlevel, and a protein activity. In another alternative embodiment, themeasured attributes are selected from the group consisting of amutation, a differential genetic sequence object, a gene copy number, atranscription level, a translation level, a protein activity, and aprotein interaction. In a preferred embodiment, the pathway is within aregulatory pathway network. In a more preferred embodiment, theregulatory pathway network is selected from the group consisting of anageing pathway network, an apoptosis pathway network, a homeostasispathway network, a metabolic pathway network, a replication pathwaynetwork, and an immune response pathway network. In a yet more preferredembodiment, the pathway is within a signaling pathway network. In analternative yet more preferred embodiment, the pathway is within anetwork of distinct pathway networks. In a most preferred embodiment,the signaling pathway network is selected from the group consisting of acalcium/calmodulin dependent signaling pathway network, a cytokinemediated signaling pathway network, a chemokine mediated signalingpathway network, a growth factor signaling pathway network, a hormonesignaling pathway network, a MAP kinase signaling pathway network, aphosphatase mediated signaling pathway network, a Ras superfamilymediated signaling pathway network, and a transcription factor mediatedsignaling pathway network.

The invention also provides a method of generating a dynamic pathway map(DPM), the method comprising: providing access to a model database thatstores a probabilistic pathway model that comprises a plurality ofpathway elements; wherein a first number of the plurality of pathwayelements are cross-correlated and assigned an influence level for atleast one pathway on the basis of known attributes; wherein a secondnumber of the plurality of pathway elements are cross-correlated andassigned an influence level for at least one pathway on the basis ofassumed attributes; and using a plurality of measured attributes for aplurality of elements of a patient sample, via an analysis engine, tomodify the probabilistic pathway model to obtain the DPM, wherein theDPM has reference pathway activity information for a particular pathway.

In one preferred embodiment, the pathway is within a regulatory pathwaynetwork, a signaling pathway network, or a network of distinct pathwaynetworks. In another preferred embodiment, the pathway element is aprotein selected from the group consisting of a receptor, a hormonebinding protein, a kinase, a transcription factor, a methylase, ahistone acetylase, and a histone deacetylase or a nucleic acid isselected from the group consisting of a genomic regulatory sequence, aregulatory RNA, and a trans-activating sequence. In a still furtherpreferred embodiment, the reference pathway activity information isspecific with respect to a normal tissue, a diseased tissue, an ageingtissue, or a recovering tissue. In another preferred embodiment, theknown attribute is selected from the group consisting of a compoundattribute, a class attribute, a gene copy number, a transcription level,a translation level, and a protein activity. In another preferredembodiment, the assumed attribute is selected from the group consistingof a compound attribute, a class attribute, a gene copy number, atranscription level, a translation level, and a protein activity. In astill further preferred embodiment, the measured attributes are selectedfrom the group consisting of a mutation, a differential genetic sequenceobject, a gene copy number, a transcription level, a translation level,a protein activity, and a protein interaction.

The invention further provides a method of analyzing biologicallyrelevant information, comprising: providing access to a model databasethat stores a dynamic pathway map (DPM), wherein the DPM is generated bymodification of a probabilistic pathway model with a plurality ofmeasured attributes for a plurality of elements of a first cell orpatient sample; obtaining a plurality of measured attributes for aplurality of elements of a second cell or patient sample; and using theDPM and the plurality of measured attributes for the plurality ofelements of the second cell or patient sample, via an analysis engine,to determine a predicted pathway activity information for the secondcell or patient sample. In one preferred embodiment, the measuredattributes for the plurality of elements of the first cell or patientsample are characteristic for a healthy cell or tissue, a specific ageof a cell or tissue, a specific disease of a cell or tissue, a specificdisease stage of a diseased cell or tissue, a specific gender, aspecific ethnic group, a specific occupational group, and a specificspecies. In another preferred embodiment, the measured attributes forthe plurality of elements of the second cell or patient sample areselected from the group consisting of a mutation, a differential geneticsequence object, a gene copy number, a transcription level, atranslation level, a protein activity, and a protein interaction. In analternative preferred embodiment, the first and second samples areobtained from the same cell or patient, and further comprising providinga treatment to the cell or patient before obtaining the plurality ofmeasured attributes for the plurality of elements of the second cell orpatient sample. In a more preferred embodiment, the treatment isselected from the group consisting of radiation, administration of apharmaceutical to the patient, and administration of a candidatemolecule to the cell. In another more preferred embodiment, thecandidate molecule is a member of a library of candidate molecules. Inanother preferred embodiment, the predicted pathway activity informationidentifies an element as a hierarchical-dominant element in at least onepathway. In a more preferred embodiment, the predicted pathway activityinformation identifies an element as a disease-determinant element in atleast one pathway with respect to a disease. In an alternativeembodiment, the method further comprises a step of generating agraphical representation of predicted pathway activity information. Inan alternative embodiment, the method further comprises a step ofgenerating a treatment recommendation that is at least in part based onthe predicted pathway activity information. In an alternativeembodiment, the method further comprises a step of using the predictedpathway activity information to formulate a diagnosis, a prognosis for adisease, or a recommendation selected from the group consisting of aselection of a treatment option, and a dietary guidance. In analternative embodiment, the method further comprises a step of using thepredicted pathway activity information to identify an epigenetic factor,a stress adaptation, a state of an organism, and a state of repair orhealing.

In another embodiment, The invention provides a transformation methodfor creating a matrix of integrated pathway activities (IPAs) forpredicting a clinical outcome for an individual in need, the methodcomprising the steps of (i) providing a set of curated pathways, whereinthe pathways comprise a plurality of entities; (ii) converting eachcurated pathway into a distinct probabilistic graphical model (PGM),wherein the PGM is derived from factor graphs of each curated pathway,(iii) providing a biological sample from the individual wherein thebiological sample comprises at least one endogenous entity comprised inone of the curated pathways; (iv) determining the levels of endogenousentity in the biological sample; (v) comparing the levels of theendogenous entity with those levels of the entity in a previouslydetermined control sample from another individual; (vi) determiningwhether the levels of the endogenous entity relative to the controlentity levels are activated, nominal, or inactivated; (vii) assigningthe endogenous entity a numeric state, wherein the state representingactivated is +1, the state representing nominal activity is 0, andwherein the state representing inactivated is −1; (viii) repeating stepsii through (vi) for another endogenous entity; (x) compiling the numericstates of each endogenous entity into a matrix of integrated pathwayactivities (IPAs), (x) wherein the matrix of integrated pathwayactivities is A wherein A, represents the inferred activity of entity iin biological sample j; the method resulting in a matrix of integratedpathway activities for predicting a clinical outcome for the individual.

In one embodiment the method for creating a matrix of IPAs comprisespredicting a clinical outcome, providing a diagnosis, providing atreatment, delivering a treatment, administering a treatment, conductinga treatment, managing a treatment, or dispensing a treatment to anindividual in need. In another embodiment, the set of curated pathwaysis from an analysis of human biology. In yet another alternativeembodiment, the set of curated pathways is from an analysis of non-humanbiology. In another embodiment, the determining of the levels of theendogenous entity relative to the control entity levels is performedusing Student's t-test. In an alternative embodiment, the determining ofthe levels of the endogenous entity relative to the control entitylevels is performed using ANOVA. In another embodiment, the transformingmethod comprise the steps of wherein a plurality of matrices ofintegrated pathway activities from more than one individual arecombined, the combined plurality of matrices resulting in a cluster, andwhere the distances between the individuals' matrices of the resultingcluster are determined. In one embodiment, the determined distances areanalysed using K-means cluster analysis. In another alternativeembodiment, the determined distances are analysed using K²-means clusteranalysis. In a yet other embodiment, the transforming method comprisesthe step of determining the levels of endogenous entity in thebiological sample comprises detecting the endogenous entity with anantibody and thereby determining the levels of endogenous entity. In analternative embodiment the step of determining the levels of endogenousentity in the biological sample comprises detecting the endogenousentity with a nucleic acid probe and thereby determining the levels ofendogenous entity. In another alternative embodiment, the step ofdetermining the levels of endogenous entity in the biological samplecomprises detecting the endogenous entity with an organic reagent,wherein the organic reagent binds to the endogenous entity therebyresulting in a detectable signal and thereby determining the levels ofendogenous entity.

In a still further alternative embodiment, the step of determining thelevels of endogenous entity in the biological sample comprises detectingthe endogenous entity with an inorganic reagent, wherein the inorganicreagent binds to the endogenous entity thereby resulting in a detectablesignal and thereby determining the levels of endogenous entity. Inanother alternative embodiment, the step of determining the levels ofendogenous entity in the biological sample comprises detecting theendogenous entity with an organic reagent, wherein the organic reagentreacts with the endogenous entity thereby resulting in a detectablesignal and thereby determining the levels of endogenous entity. Inanother alternative embodiment, the step of determining the levels ofendogenous entity in the biological sample comprises detecting theendogenous entity with an inorganic reagent, wherein the inorganicreagent reacts with the endogenous entity thereby resulting in adetectable signal and thereby determining the levels of endogenousentity. In a preferred embodiment, the step of determining the levels ofendogenous entity in the biological sample comprises measuring theabsorbance of the endogenous entity at the optimal wavelength for theendogenous entity and thereby determining the levels of endogenousentity. In an alternative preferred embodiment, the step of determiningthe levels of endogenous entity in the biological sample comprisesmeasuring the fluorescence of the endogenous entity at the optimalwavelength for the endogenous entity and thereby determining the levelsof endogenous entity. In a still further alternative preferredembodiment, the step of determining the levels of endogenous entity inthe biological sample comprises reacting the endogenous entity with anenzyme, wherein the enzyme selectively digests the endogenous entity tocreate at least one product, detecting the at least one product, andthereby determining the levels of endogenous entity. In a more preferredembodiment, the step of reacting the endogenous entity with an enzymeresults in creating at least two products. In a yet more preferredembodiment, the step of reacting the endogenous entity with an enzymeresulting at least two products is followed by a step of treating theproducts with another enzyme, wherein the enzyme selectively digests atleast one of the products to create at least a third product, detectingthe at least a third product, and thereby determining the levels ofendogenous entity.

In another preferred embodiment the individual is selected from thegroup of a healthy individual, an asymptomatic individual, and asymptomatic individual. In a more preferred embodiment, the individualis selected from the group consisting of an individual diagnosed with acondition, the condition selected from the group consisting of a diseaseand a disorder. In a preferred embodiment, the condition is selectedfrom the group consisting of acquired immunodeficiency syndrome (AIDS),Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, benign prostatichyperplasia, bronchitis, Chediak-Higashi syndrome, cholecystitis,Crohn's disease, atopic dermatitis, dermnatomyositis, diabetes mellitus,emphysema, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, chronicgranulomatous diseases, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polycystic ovary syndrome,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, severe combined immunodeficiency disease (SCID), Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infection; and adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and utenis, akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis (ALS),ataxias, bipolar disorder, catatonia, cerebral palsy, cerebrovasculardisease Creutzfeldt-Jakob disease, dementia, depression, Down'ssyndrome, tardive dyskinesia, dystonias, epilepsy, Huntington's disease,multiple sclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain. In an alternativepreferred embodiment, the condition is selected from the groupconsisting of cancers such as adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus; immunedisorders such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and developmental disorders such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, for example, the brain, adrenal gland, kidney, skeletal orreproductive system. In another preferred embodiment, the condition isselected from the group consisting of endocrinological disorders such asdisorders associated with hypopituitarism including hypogonadism,Sheehan syndrome, diabetes insipidus, Kaftan's disease,Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis,empty sella syndrome, and dwarfism; hyperpituitarism includingacromegaly, giantism, and syndrome of inappropriate antidiuretic hormone(ADH) secretion (SIADH); and disorders associated with hypothyroidismincluding goiter, myxedema, acute thyroiditis associated with bacterialinfection, subacute thyroiditis associated with viral infection,autoimmune thyroiditis (Hashimoto's disease), and cretinism; disordersassociated with hyperthyroidism including thyrotoxicosis and its variousforms, Grave's disease, pretibial myxedema, toxic multinodular goiter,thyroid carcinoma, and Plummer's disease; and disorders associated withhyperparathyroidism including Conn disease (chronic hypercalcmia);respiratory disorders such as allergy, asthma, acute and chronicinflammatory lung diseases, ARDS, emphysema, pulmonary congestion andedema, COPD, interstitial lung diseases, and lung cancers; cancer suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immunological disorderssuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma.

The Invention also provides the transforming method as disclosed hereinwherein matrix A can then be used in place of the original constituentdatasets to identify associations with clinical outcomes. In a morepreferred embodiment the curated pathways are selected from the groupconsisting of biochemical pathways, genetic pathways, metabolicpathways, gene regulatory pathways, gene transcription pathways, genetranslation pathways. In another more preferred embodiment, the entitiesare selected from the group consisting of nucleic acids, peptides,proteins, peptide nucleic acids, carbohydrates, lipids, proteoglycans,factors, co-factors, biochemical metabolites, organic compositions,inorganic compositions, and salts. In a yet other preferred embodiment,the biological sample is selected from the group consisting of patientsamples, control samples, experimentally-treated animal samples,experimentally-treated tissue culture samples, experimentally-treatedcell culture samples, and experimentally-treated in vitro biochemicalcomposition samples. In a more preferred embodiment, the biologicalsample is a patient sample.

The invention also provides a probabilistic graphical model (PGM)framework having an output that infers the molecular pathways altered ina patient sample, the PGM comprising a plurality of factor graphs,wherein the factor graphs represent integrated biological datasets, andwherein the inferred molecular pathways that are altered in a patientsample comprise molecular pathways known from data and wherein saidmolecular pathways effect a clinical or non-clinical condition, whereinthe inferred molecular pathways are known to be modulated by a clinicalregimen or treatment, and wherein the output indicates a clinicalregimen. In a preferred embodiment, the data is selected fromexperimental data, clinical data, epidemiological data, andphenomenological data. In another preferred embodiment, the condition isselected from the group consisting of a disease and a disorder. In amore preferred embodiment, the condition is selected from the groupconsisting of acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, benign prostatic hyperplasia,bronchitis, Chediak-Higashi syndrome, cholecystitis, Crohn's disease,atopic dermatitis, dermnatomyositis, diabetes mellitus, emphysema,erythroblastosis fetalis, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, chronic granulomatousdiseases, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polycystic ovary syndrome, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, severe combinedimmunodeficiency disease (SCID), Sjogren's syndrome, systemicanaphylaxis, systemic lupus erythematosus, systemic sclerosis,thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,complications of cancer, hemodialysis, and extracorporeal circulation,viral, bacterial, fungal, parasitic, protozoal, and helminthicinfection; and adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus, akathesia. Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis (ALS), ataxias, bipolardisorder, catatonia, cerebral palsy, cerebrovascular diseaseCreutzfeldt-Jakob disease, dementia, depression, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain. In an alternative morepreferred embodiment, the condition is selected from the groupconsisting of cancers such as adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus; immunedisorders such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and developmental disorders such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, for example, the brain, adrenal gland, kidney, skeletal orreproductive system. In a yet other more preferred embodiment, thecondition is selected from the group consisting of endocrinologicaldisorders such as disorders associated with hypopituitarism includinghypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease,Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis,empty sella syndrome, and dwarfism; hyperpituitarism includingacromegaly, giantism, and syndrome of inappropriate antidiuretic hormone(ADH) secretion (SIADH); and disorders associated with hypothyroidismincluding goiter, myxedema, acute thyroiditis associated with bacterialinfection, subacute thyroiditis associated with viral infection,autoimmune thyroiditis (Hashimoto's disease), and cretinism; disordersassociated with hyperthyroidism including thyrotoxicosis and its variousforms, Grave's disease, pretibial myxedema, toxic multinodular goiter,thyroid carcinoma, and Plummer's disease; and disorders associated withhyperparathyroidism including Conn disease (chronic hypercalemia);respiratory disorders such as allergy, asthma, acute and chronicinflammatory lung diseases, ARDS, emphysema, pulmonary congestion andedema, COPD, interstitial lung diseases, and lung cancers; cancer suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immunological disorderssuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an overview of the PARADIGM method. PARADIGM uses apathway schematic with functional genomic data to infer geneticactivities that can be used for further downstream analysis. NCI Pathwayinteractions in TCGA GBM data. For all (n=462) pairs where A was foundto be an upstream activator of gene B in NCI-Nature Pathway Database,the Pearson correlation (x-axis) computed from the TCGA GBM data wascalculated in two different ways. The histogram plots the correlationsbetween the A's copy number and B's expression (C2E, solid red) andbetween A's expression and B's expression (E2E, solid blue). A histogramof correlations between randomly paired genes is shown for C2E (dashedred) and E2E (dashed blue). Arrows point to the enrichment of positivecorrelations found for the C2E (red) and E2E (blue) correlation

FIGS. 2A-2C illustrate the conversion of a genetic pathway diagram intoa PARADIGM model. Overview of the PARADIGM method. PARADIGM uses apathway schematic with functional genomic data to infer geneticactivities that can be used for further downstream analysis. FIG. 2AData on a single patient is integrated for a single gene using a set offour different biological entities for the gene describing the DNAcopies, mRNA and protein levels, and activity of the protein. FIG. 2BPARADIGM models various types of interactions across genes includingtranscription factors to targets' (upper-left), subunits aggregating ina complex (upper-right), post-translational modification (lower-left),and sets of genes in a family performing redundant functions(lower-right). FIG. 2C Toy example of a small sub-pathway involving P53,an inhibitor MDM2, and the high level process, apoptosis as representedin the model.

FIG. 3 illustrates exemplary NCI pathway interactions in The CancerGenome Atlas (TCGA) project (cancergerionic.nih.gov) glioblastomamultiform (GMB) data. For all (n=462) pairs where A was found to be anupsterama ctivator of gene B in NCI-Nature Pathway Database, the Pearsoncorrelation (x-axis) computed from the TCGA GMB data was calculated intwo different ways. The histogram plots the correlations between the A'scopy number and B's expression (C2E, solid red) and between A'sexpression and B's expression (E2E, solid blue). A histogram ofcorrelations between randomly paired genes is shown for C2E (dashed red)and E2E (dashed blue). Arrows point to the enrichment of positivecorrelations found for the C2E (red) and E2E (blue) correlation.

FIG. 4 illustrates exemplary learning parameters for the anti-apoptoticserine-threonine kinase 1 (AKTI). Integrated Pathway Activities (IPAs)are shown at each iteration of the Expectation-Maximization (EM)algorithm until convergence. Dots show IPAs from permuted samples andcircles show IPAs fdrom real samples. The red line denotes the mean IPAin real samples and the green line denotes the man IPA of null samples.

FIG. 5 illustrates distinguishing decoy from real pathways with PARADIGMand Signaling Pathway Impact Analysis (SPIA). Decoy pathways werecreated by assigning a new gene name to each gene in a pathway. PARADIGMand SPIA were then used to compute the perturbation of every pathway.Each line shows the receiver-operator characteristic for distinguishingreal from decoy pathways using the perturbation ranking. In breastcancer, for example, the areas under the curve (AUCs) are 0.669 and0.602 for PARADIGM and SPIA, respectively. In glioblastoma multiform(GBM), the AUCs are 0.642 and 0.604, respectively.

FIG. 6 illustrates exemplary patient sample IPAs compared with withinpermutations for Class I phosphatidylinositol-3-kinase (PI3K) signalingevents mediated by Akt in breast cancer.

Biological entities were sorted by mean IPA in the patient samples (red)and compared with the mean IPA for the peruted samples. The coloredareas around each mean denote the standard deviation (SD) of each set.The IPAs of the right include AKT1, CHUK, and MDM2.

FIG. 7 illustrates an exemplary CIRCLEMAP display of the ErbB2 pathway.For each node, estrogen receptor (ER) status, IPAs, expression data, andcopy-number data are displayed as concentric circles, from innermost tooutermost respectively. The apoptosis node and theErbB2/ErbB3/neuregulin 2 complex node have circles only for ER statusand for IPAs, as there are no direct observations of these entities.Each patient's data is displayed along one angle from the circle centerto edge.

FIG. 8 illustrates exemplary clustering of IPAs for TCGA GBM. Eachcolumn corresponds to a single sample, and each row to a biomolecularentity. Color bars beneath the hierarchical clustering tree denoteclusters used for FIG. 9.

FIG. 9 illustrates Kaplan-Meier survival plots for the clusters fromFIG. 8.

FIGS. 10A-10D illustrate that cell lines show a broad range of responsesto therapeutic compounds. FIG. 10A Luminal and ERBB2AMP cell linespreferentially respond to AKT inhibition. Each bar represents theresponse of a single breast cancer cell line to the Sigma AKT1-2inhibitor. Cell lines are ordered by increasing sensitivity(−log₁₀(GI₅₀)) and colored according to subtype. FIG. 10B GI₅₀ valuesfor compounds with similar mechanisms are highly correlated. Heatmapshows hierarchical clustering of correlations between responses breastcancer cell lines treated with various compounds. FIG. 10C Compoundswith similar modes of action show similar patterns of response acrossthe panel of cell lines. Each column represents one cell line, each rowrepresents a compound tested. 0150 values are hierarchically clustered.Only compounds with a significant subtype effect are included. Celllines of similar subtype tend to cluster together, indicating that theyare responsive to the same compounds. Gray represents missing values.FIG. 10D CNAs are associated sensitivity. Boxplots show distribution ofresponse sensitivity for cell lines with aberrant (A) and normal (N)copy number at the noted genomic locus. FDR p values for the associationbetween drug response and CNA are noted. a. 9p21 (CDKN2A) deletion isassociated with response to ixabepilone, vinerolbine and fascaplysin. b.20q13 (STK15/AURKA) amplification is associated with VX-680 andGSK1070916. c. Amplification at 11q13 (CCND1) is associated withresponse to carboplatin and GSK1070916.

FIG. 11 shows a heatmap of non-redundant PARADIGM activities both cellline and TCGA samples. Cluster dendrogram represents Euclidian distancebetween samples and was created using Eisen Cluster and drawn using JavaTreeview. Colored bars below dendrogram represent sample subtype (top)and sample cohort (bottom).

FIGS. 12A-12D illustrate that cell line subtypcs have unique networkfeatures. In all panels, each node in the graph represents a differentpathway “concept” corresponding to either a protein (circles), amultimeric complex (hexagons), or a an abstract cellular process(squares). The size of the nodes were drawn in proportion to thedifferential activity score such that larger nodes correspond to pathwayconcepts with activities more correlated with basal versus non-basalcell lines. Color indicates whether the concept is positively correlated(red) or negatively correlated (blue) with the basal subtype. Linksrepresent different interactions including protein-protein levelinteractions (dashed lines) and transcriptional (solid lines).Interactions were included in the map only if they interconnect conceptswhose absolute level of differential activity is higher than the meanabsolute level. FIG. 12A The MYC/MAX and ERK1/2 subnet is preferentiallyactivated in basal breast cancer cell lines. FIG. 12B The CTTNB Inetwork is activated in claudin-low cell lines. FIG. 12C A FOXA1/FOXA2network is upregulated in the luminal subtype. FIG. 12D The ERBB2AMPsubtype shows down-regulation of the RPS6KB1 pathway.

FIGS. 13A-13B illustrate how pathway diagrams can be used to predictresponse to therapies. FIG. 13A Upper panel. Basal breast cancer celllines preferentially respond to the DNA damaging agent cisplatin. Lowerpanel. Basal cell lines show enhanced activity in pathways associatedwith the DNA damage response, providing a possible mechanism by whichcisplatin acts in these cell lines. FIG. 13B Upper panel. ERBB2AMP celllines are sensitive to the HSP90 inhibitor geldanamycin. Lower panel.The ERBB2-HSP90 network is upregulated in ERBBP2AMP cell lines.Convention as in FIG. 3 12.

FIGS. 14A-14B illustrates exemplary genomic and transcriptional profilesof the breast cancer cell lines. FIG. 14A DNA copy number aberrationsfor 43 breast cancer cell lines are plotted with log₁₀(FDR) of GISTICanalysis on the y-axis and chromosome position on the x-axis. Copynumber gains are shown in red with positive log₁₀(FDR) and losses areshown in green with negative log₁₀(FDR). FIG. 14B Hierarchical concensusclustering matrix for 55 breast cancer cell lines showing 3 clusters(claudin-low, luminal, basal) based on gene expression signatures. Foreach cell line combination, color intensity is proportional toconsensus.

FIGS. 15A-15B illustrate that GI₅₀ calculations are highly reproducible.FIG. 15A Each bar a count of the frequency of replicated drug/cell linecombinations. Most cell lines were tested only one time against aparticular compound, but some drug/cell line combinations were testedmultiple times. FIG. 15B Each boxplot represents the distribution ofmedian average deviations for drug/cell line pairs with 3 or 4replicates.

FIGS. 16A-16B show that doubling time varies across cell line subtype.FIG. 16A Growth rate, computed as the median doubling time in hours, ofthe breast cancer cell lines subtypes are shown as boxplots. The basaland claudin-low subtypes have shorter median doubling time as comparedto lumina (and ERBB2^(AmP) subtypes, Kruskal-Wallis p value (p=0.006).FIG. 16B The ANCOVA model shows strong effects of both subtype andgrowth rate on response to 5′FU. Luminal (black) and basal/claudin-low(red) breast cancer lines each show significant associations to growthrate but have distinct slopes.

FIG. 17 shows that inferred pathway activities are more stronglycorrelated within subtypes than within cohorts. Shown is a histogram oft-statistics derived from Pearson correlations computed between celllines and TCGA samples of the same subtype (red) compared tot-statistics of Pearson correlations between cell lines of differentsubtypes (black). X-axis corresponds to the Pearson correlationt-statistic; y-axis shows the density of (cell-line, cell-line) or(cell-line, TCGA sample) pairs. K-S test (P<1×10⁻²²) indicates celllines and TCGA samples of the same subtype are more alike than celllines of other subtypes.

Supplementary FIGS. 18-21 illustrate an exemplary network architecturefor each of the four subnetworks identified from the SuperPathway.

FIG. 18 illustrates a network diagram of basal pathway markers. Eachnode in the graph represents a different pathway “concept” correspondingto either a protein (circles), a multimeric complex (hexagons), or a anabstract cellular process (squares). The size of the nodes are drawn inproportion to the differential activity score such that larger nodescorrespond to pathway concepts with activities more correlated withbasal versus non-basal cell lines. Color indicates whether the conceptis positively correlated (red) or negatively correlated (blue) with thebasal subtype. Links represent different interactions includingprotein-protein level interactions (dashed lines) and transcriptional(solid lines). Interactions were included in the map only if theyinterconnect concepts whose absolute level of differential activity ishigher than the mean absolute level.

FIG. 19 illustrates an exemplary network diagram of claudin-low pathwaymarkers. Convention as in FIG. 18.

FIG. 20 illustrates an exemplary network diagram of luminal pathwaymarkers. Convention as in FIG. 18.

FIG. 21 illustrates an exemplary network diagram of ERBB2AMP pathwaymarkers. Convention as in FIG. 18.

FIGS. 22A-22C illustrates an exemplary URICB-FOXM1-CCNB1 networks inluminal, claudin-low and basal cell lines. FIG. 22A Network surroundingAURKB and FOXM1 in luminal cell lines. CCNB1 was not significantlydownregulated and therefore does not appear on the pathway map. FIG. 22BIn claudin-low cell lines, AURKB and FOXM1 both up-regulated; activityfor CCNB1 was not significant. FIG. 22C AURKB, FOXM1 and CCNB1 are allup-regulated in basal cell lines. Convention as in FIG. 18.

FIG. 23 illustrates an exemplary distribution of unsupervised clustersand survival curves of the patients of the MicMa cohort according toCNA, mRNA expression, DNA methylation and miRNA expression. For eachtype of genomic level the size of each cluster are plotted on the left,and to the right, survival curves are shown. Significance ofdifferential survival are assessed by two methods (see Examples).

FIG. 24 illustrates an exemplary distribution of indentified PARADIGMclusters and survival. Each bar represents the size of each cluster.Heatmap of Paradigm IPLs for the MicMa dataset. Survival curves of theMicMa Paradigm clusters after mapping to the Chin-Naderi-Caldasdatasets.

FIG. 25 illustrates an exemplary heatmaps of Paradigm IPLs for eachdataset. Each row shows the IPL of a gene or complex across all threecohorts. The colored bar across the top shows the MicMa-derived Paradigmclusters, as in FIG. 2. Members of pathways of interest are labeled bytheir pathway. Red represents an activated IPL, blue a deactivated IPL.

FIG. 26 illustrates the FOXM1 Transcription Factor Network. The uppernetwork diagram summarizes data from cluster pdgm 0.3, whereas the lowercluster summarizes the data from other clusters. Nodes shapes denote thedata type which was most frequently perturbed within each cluster, andnode color denote the direction of perturbation. Edge arrows denote thesign of interactions, and color denotes the type of interaction.

FIG. 27 illustrates a toy example of a small fragment of the p53apoptosis pathway. A pathway diagram from NCI was converted into afactor graph that includes both hidden and observed states.

FIG. 28 illustrates an exemplary heatmap of Inferred Pathway Activities(IPAs). IPAs representing 1598 inferences of molecular entities (rows)inferred to be activated (red) or inactivated (blue) are plotted foreach of 316 patient tumor samples (columns). IPAs were hierarchicallyclustered by pathway entity and tumor sample, and labels on the rightshow sections of the heatmap enriched with entities of individualpathways. The colorbar legend is in log base 10.

FIG. 29 summarises FOXM1 integrated pathway activities (IPAs) across allsamples. The arithmetic mean of IPAs across tumor samples for eachentity in the FOXM1 transcription factor network is shown in red, withheavier red shading indicating two standard deviations. Gray line andshading indicates the mean and two standard deviations for IPAs derivedfrom the 1000 “null” samples.

FIGS. 30A-30B show a comparison of IPAs of FOXM1 to those of othertested transcription factors (TFs) in NCI Pathway Interaction Database.FIG. 30A Histogram of IPAs with non-active (zero-valued) IPAs removed.FOXM1 targets are significantly more activated than other NCI TFs (P<₁₀²⁶⁷, Kolmogorov-Smirnov (KS) test). FIG. 30B Histogram of all IPAsincluding non-active IPAs. Using all IPAs, FOXM1's activity relative toother TFs is interpreted with somewhat higher significance (P<10⁻³¹; KStest).

FIG. 31 illustrates that FOXM1 is not expressed in fallopian epitheliumcompared to serous ovarian carcinoma. FOXM1's expression levels infallopian tube was compared to its levels in serous ovarian carcinomausing the data from Tone et al (PMID: 18593983). FOXM1's expression ismuch lower in fallopian tube, including in samples carrying BRCA1/2mutations, indicating that FOXM1's elevated expression observed in theTCGA serous ovarian cancers is not simply due to an epithelialsignature.

FIG. 32 shows expression of FOXM1 transcription factor network genes inhigh grade versus low grade carcinoma. Expression levels for FOXM1 andnine selected FOXM1 targets (based on NCI-PID) were plotted for bothlow-grade (I; tan boxes; 26 samples) and high-grade (II/III; blue boxes;296 samples) ovarian carcinomas. Seven out of the nine targets wereshowed to have significantly high expression of FOXM1 in the high-gradecarcinomas (Student's t-test; p-values noted under boxplots). CDKN2A mayalso be differentially expressed but had a borderline t-statistic(P=0.01). XRCC I was detected as differentially expressed.

FIGS. 33A-33D show that the cell lines show a broad range of responsesto therapeutic compounds. FIG. 33A Luminal and ERBB2AMP cell linespreferentially respond to AKT inhibition. Each bar represents theresponse of a single breast cancer cell line to the Sigma AKT1-2inhibitor. Cell lines are ordered by increasing sensitivity(−log₁₀(GI₅₀)) and colored according to subtype. FIG. 33B GI₅₀ valuesfor compounds with similar mechanisms are highly correlated. Heatmapshows hierarchical clustering of correlations between responses breastcancer cell lines treated with various compounds. FIG. 33C Compoundswith similar modes of action show similar patterns of response acrossthe panel of cell lines. Each column represents one cell line, each rowrepresents a compound tested. GI₅₀ values are hierarchically clustered.Only compounds with a significant subtype effect are included. Celllines of similar subtype tend to cluster together, indicating that theyare responsive to the same compounds. Gray represents missing values.FIG. 33D CNAs are associated sensitivity. Boxplots show distribution ofresponse sensitivity for cell lines with aberrant (A) and normal (N)copy number at the noted genomic locus. FDR p values for the associationbetween drug response and CNA are noted. a. 9p2I (CDKN2A) deletion isassociated with response to ixabepilone, vinerolbine and fascaplysin. b.20q13 (STK15/AURKA) amplification is associated with VX-680 andGSK1070916. c. Amplification at 11q13 (CCND I) is associated withresponse to carboplatin and GSK1070916.

FIG. 34 Heatmap of non-redundant PARADIGM activities both cell line andTCGA samples. Cluster dendrogram represents Euclidian distance betweensamples and was created using Eisen Cluster and drawn using JavaTreeview. Colored bars below dendrogram represent sample subtype (top)and sample cohort (bottom).

FIGS. 35A-35D show that the cell line subtypes have unique networkfeatures. In all panels, each node in the graph represents a differentpathway “concept” corresponding to either a protein (circles), amultimeric complex (hexagons), or a an abstract cellular process(squares). The size of the nodes were drawn in proportion to thedifferential activity score such that larger nodes correspond to pathwayconcepts with activities more correlated with basal versus non-basalcell lines. Color indicates whether the concept is positively correlated(red) or negatively correlated (blue) with the basal subtype. Linksrepresent different interactions including protein-protein levelinteractions (dashed lines) and transcriptional (solid lines).Interactions were included in the map only if they interconnect conceptswhose absolute level of differential activity is higher than the meanabsolute level. FIG. 35A The MYC/MAX and ERK1/2 subnet is preferentiallyactivated in basal breast cancer cell lines. FIG. 35B The C′FTNB1network is activated in claudin-low cell lines. FIG. 35C A FOXA1/FOXA2network is upregulated in the luminal subtype. FIG. 35D The ERBB2AMPsubtype shows down-regulation of the RPS6KB1 pathway.

FIGS. 36A-36B show that the pathway diagrams can be used to predictresponse to therapies. FIG. 36A Upper panel. Basal breast cancer celllines preferentially respond to the DNA damaging agent cisplatin. Lowerpanel. Basal cell lines show enhanced activity in pathways associatedwith the DNA damage response, providing a possible mechanism by whichcisplatin acts in these cell lines. FIG. 36B Upper panel. ERBB2AMP celllines are sensitive to the HSP90 inhibitor geldanamycin. Lower panel.The ERBB2-HSP90 network is upregulated in ERBBP2AMP cell lines.Convention as in FIG. 3 36.

FIGS. 37A-37C illustrate genome copy number abnormalities. FIG. 37ACopy-number profiles of 489 HGS-OvCa, compared to profiles of 197glioblastoma multiforme (GBM) tumors46. Copy number increases (red) anddecreases (blue) are plotted as a function of distance along the normalgenome. FIG. 37B Significant, focally amplified (red) and deleted (blue)regions are plotted along the gnome. Annotations include the 20 mostsignificant amplified and deleted regions, well-localized regions with 8or fewer genes, and regions with known cancer genes or genes identifiedby genome-wide loss-of-function screens. The number of genes included ineach region is given in brackets. FIG. 37C Significantly amplified (red)and deleted (blue) chromosome arms.

FIGS. 38A-38E illustrate gene and miRNA expression patterns of molecularsubtype and outcome prediction in HGS-OvCa. FIG. 38A Tumors from TCGAand Tothill et al. separated into four clusters, based on geneexpression. FIG. 38B Using a training dataset, a prognostic genesignature was defined and applied to a test dataset. FIG. 38CKaplan-Meier analysis of four independent expression profile datasets,comparing survival for predicted higher risk versus lower risk patients.Univariate Cox p-value for risk index included.

FIG. 38D Tumors separated into three clusters, based on miRNAexpression, overlapping with gene-based clusters as indicated. FIG. 38EDifferences in patient survival among the three miRNA-based clusters.

FIGS. 39A-39D illustrate altered Pathways in HGS-OvCa. FIG. 39A The RBand PI3K/RAS pathways, identified by curatcd analysis and FIG. 39B NOTCHpathway, identified by HotNet analysis, are commonly altered.Alterations are defined by somatic mutations, DNA copy-number changes,or in some cases by significant up- or down-regulation compared toexpression in diploid tumors. Alteration frequencies are in percentageof all cases; activated genes are red, inactivated genes are blue. FIG.39C Genes in the HR pathway are altered in up to 49% of cases. Survivalanalysis of BRCA status shows divergent outcome for BRCA mutated cases(exhibiting better overall survival) than BRCA wild-type, andBRCA/epigenetically silenced cases exhibiting worse survival. FIG. 39DThe FOXM1 transcription factor network is activated in 87% of cases.Each gene is depicted as a multi-ring circle in which its copy number(outer ring) and gene expression (inner ring) are plotted such that each“spoke” in the ring represents a single patient sample, with samplessorted in increasing order of FOXAV expression. Excitatory (red arrows)and inhibitory interactions (blue lines) were taken from the NCI PathwayInteraction Database. Dashed lines indicate transcriptional regulation.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments disclosed in this document are illustrative andexemplary and are not meant to limit the invention. Other embodimentscan be utilized and structural changes can be made without departingfrom the scope of the claims of the present invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “an miRNA” includes aplurality of such miRNAs, and a reference to “a phannaceutical carrier”is a reference to one or more phannaceutical carriers and equivalentsthereof; and so forth.

As used herein, the term “curated” means the relationships between a setof biological molecules and/or non-biological molecules that has beentested, analyzed, and identified according to scientific and/or clinicalprinciples using methods well known in the art, such as molecularbiological, biochemical, physiological, anatomical, genomic,transcriptomic, proteomic, metabolomic, ADME, and bioinformatictechniques, and the like. The relationships may be biochemical such asbiochemical pathways, genetic pathways, metabolic pathways, generegulatory pathways, gene transcription pathways, gene translationpathways, miRNA-regulated pathways, pseudogene-regulated pathways, andthe like.

High-throughput data is providing a comprehensive view of the molecularchanges in cancer tissues. New technologies allow for the simultaneousgenome-wide assay of the state of genome copy number variation, geneexpression, DNA methylation, and epigenetics of tumor samples and cancercell lines.

Studies such as The Cancer Genome Atlas (TCGA), Stand Up To Cancer(SU2C), and many more are planned in the near future for a wide varietyof tumors. Analyses of current data sets find that genetic alterationsbetween patients can differ but often involve common pathways. It istherefore critical to identify relevant pathways involved in cancerprogression and detect how they are altered in different patients.

We disclose a novel method for inferring patient-specific geneticactivities incorporating curated pathway interactions among genes. Agene is modeled by a factor graph as a set of interconnected variablesencoding the expression and known activity of a gene and its products,allowing the incorporation of many types of -omic data as evidence.

The method predicts the degree to which a pathway's activities (forexample, internal gene states, interactions, or high-level “outputs”)are altered in the patient using probabilistic inference. Compared to acompeting pathway activity inference approach, called SPIA, our methodidentifies altered activities in cancer-related pathways with fewerfalse-positives in, but not limited to, both a glioblastoma multiform(GBM) and a breast cancer dataset.

Pathway Recognition Algorithm using Data integration on Genomic Models(PARADIGM) identified consistent pathway-level activities for subsets ofthe GBM patients that are overlooked when genes are considered inisolation. Further, grouping GBM patients based on their significantpathway perturbations using the algorithm divides them intoclinically-relevant subgroups having significantly different survivaloutcomes.

These findings suggest that therapeutics might be chosen that can targetgenes at critical points in the commonly perturbed pathway(s) of a groupof patients or of an individual.

We describe a probabilistic graphical model (PGM) framework based onfactor graphs (Kschischang:2001 supra) that can integrate any number ofgenomic and functional genomic datasets to infer the molecular pathwaysaltered in a patient sample. We tested the model using copy numbervariation and gene expression data for both a glioblastoma and breastcancer dataset. The activities inferred using a structured pathway modelsuccessfully stratify the glioblastoma patients into clinically-relevantsubtypes. The results suggest that the pathway-informed inferences aremore informative than using gene-level data in isolation.

In addition to providing better prognostics and diagnostics, integratedpathway activations offer important clues about potential therapeuticsthat could be used to abrogate disease progression.

We developed an approach called PARADIGM (PAthway Recognition Algorithmusing Data Integration on Genomic Models) to infer the activities ofgenetic pathways from integrated patient data. FIG. 1 illustrates theoverview of the approach. Multiple genome-scale measurements on a singlepatient sample are combined to infer the activities of genes, products,and abstract process inputs and outputs for a single National CancerInstitute (NCI) pathway. PARADIGM produces a matrix of integratedpathway activities (IPAs) A where Au represents the inferred activity ofentity i in patient sample j. The matrix A can then be used in place ofthe original constituent datasets to identify associations with clinicaloutcomes.

We first converted each NCI pathway into a distinct probabilistic model.A toy example of a small fragment of the p53 apoptosis pathway is shownin FIG. 2C. A pathway diagram from NCI was converted into a factor graphthat includes both hidden and observed states (FIGS. 2A-2C). The factorgraph integrates observations on gene- and biological process-relatedstate information with a structure describing known interactions amongthe entities.

To represent a biological pathway with a factor graph, we use variablesto describe the states of entities in a cell, such as a particular mRNAor complex, and use factors to represent the interactions andinformation flow between these entities. These variables represent thedifferential state of each entity in comparison to a “control” or normallevel rather than the direct concentrations of the molecular entities.This representation allows us to model many high-throughput datasets,such as gene expression detected with DNA microarrays that often eitherdirectly measure the differential state of a gene or convert directmeasurements to measurements relative to matched controls. It alsoallows for many types of regulatory relationships among genes. Forexample, the interaction describing MDM2 mediating ubiquitin-dependentdegradation of p53 can be modeled as activated MDM2 inhibiting levels ofp53 protein.

In one embodiment, the method may be used to provide clinicalinformation that can be used in a variety of diagnostic and therapeuticapplications, such as detection of cancer tissue, staging of cancertissue, detection of metastatic tissue, and the like; detection ofneurological disorders, such as, but not limited to, Alzheimer'sdisease, amyotrophic lateral sclerosis (ALS), Parkinson's disease,schizophrenia, epilepsy, and their complications; developmentaldisorders such as DiGeorge Syndrome, autism, autoimmune disorders suchas multiple sclerosis, diabetes, and the like; treatment of aninfection, such as, but not limited to, viral infection, bacterialinfection, fungal infection, leishmania, schistosomiasis, malaria,tape-worm, elephantiasis, infections by nematodes, nematines, and thelike.

In one embodiment, the method may be used to provide clinicalinformation to detect and quantify altered gene expression,absence/presence versus excess, expression of mRNAs or to monitor mRNAlevels during therapeutic intervention. Conditions, diseases ordisorders associated with altered expression include acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, benign prostatic hyperplasia, bronchitis, Chediak-Higashisyndrome, cholecystitis, Crohn's disease, atopic dermatitis,dennnatomyositis, diabetes mellitus, emphysema, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, chronic granulomatous diseases, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polycystic ovary syndrome, polymyositis, psoriasis, Reiter's syndrome,rheumatoid arthritis, scleroderma, severe combined immunodeficiencydisease (SCID), Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infection; and adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. The diagnostic assay may use hybridization or amplificationtechnology to compare gene expression in a biological sample from apatient to standard samples in order to detect altered gene expression.Qualitative or quantitative methods for this comparison are well knownin the art.

In one embodiment, the method may be used to provide clinicalinformation to detect and quantify altered gene expression; absence,presence, or excess expression of mRNAs; or to monitor mRNA levelsduring therapeutic intervention. Disorders associated with alteredexpression include akathesia, Alzheimer's disease, amnesia, amyotrophiclateral sclerosis (ALS), ataxias, bipolar disorder, catatonia, cerebralpalsy, cerebrovascular disease Creutzfeldt-Jakob disease, dementia,depression, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, muscular dystrophy,neuralgias, neurofibromatosis, neuropathies, Parkinson's disease, Pick'sdisease, retinitis pigmentosa, schizophrenia, seasonal affectivedisorder, senile dementia, stroke, Tourette's syndrome and cancersincluding adenocarcinomas, melanomas, and teratocarcinomas, particularlyof the brain.

In one embodiment, the method may be used to provide clinicalinformation for a condition associated with altered expression oractivity of the mammalian protein. Examples of such conditions include,but are not limited to, acquired immunodeficiency syndrome (AIDS),Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, benign prostatichyperplasia, bronchitis, Chediak-Higashi syndrome, cholecystitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, chronicgranulomatous diseases, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polycystic ovary syndrome,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, severe combined immunodeficiency disease (SCID), Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infection; and adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus. akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, ataxias,bipolar disorder, catatonia, cerebral palsy, cerebrovascular diseaseCreutzfeldt-Jakob disease, dementia, depression, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain.

In one embodiment the methods disclosed herein may be used to detect,stage, diagnose, and/or treat a disorder associated with decreasedexpression or activity of the nucleic acid sequences. Examples of suchdisorders include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; immune disorders such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and developmental disorders such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, for example, the brain, adrenal gland, kidney, skeletal orreproductive system.

In one embodiment the methods disclosed herein may be used to detect,stage, diagnose, and/or treat a disorder associated with expression ofthe nucleic acid sequences. Examples of such a disorder include, but arenot limited to, endocrinological disorders such as disorders associatedwith hypopituitarism including hypogonadism, Sheehan syndrome, diabetesinsipidus, Kallman's disease, Hand-Schuller-Christian disease,Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism;hyperpituitarism including acromegaly, giantism, and syndrome ofinappropriate antidiuretic hormone (ADH) secretion (SIADH); anddisorders associated with hypothyroidism including goiter, myxedema,acute thyroiditis associated with bacterial infection, subacutethyroiditis associated with viral infection, autoimmune thyroiditis(Hashimoto's disease), and cretinism; disorders associated withhyperthyroidism including thyrotoxicosis and its various forms, Grave'sdisease, pretibial myxedema, toxic multinodular goiter, thyroidcarcinoma, and Plummer's disease; and disorders associated withhyperparathyroidism including Conn disease (chronic hypercalemia);respiratory disorders such as allergy, asthma, acute and chronicinflammatory lung diseases, ARDS, emphysema, pulmonary congestion andedema, COPD, interstitial lung diseases, and lung cancers; cancer suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immunological disorderssuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences may be used in Southern or Northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered nucleic acid sequenceexpression. Such qualitative or quantitative methods are well known inthe art.

CHARACTERIZATION AND BEST MODE OF THE INVENTION

PARADIGM: Inference of Patient-Specific Pathway Activities fromMulti-Dimensional Cancer Genomics Data Using PARADIGM.

One hypothesis of pathway-based approaches is that the geneticinteractions found in pathway databases carry information forinterpreting correlations between gene expression changes detected incancer. For example, if a cancer-related pathway includes a link from atranscriptional activator A to a target gene T, we expect the expressionof A to be positively correlated with the expression of T (E2Ecorrelation). Likewise, we also expect a positive correlation betweenA's copy number and T's expression (C2E correlation). Further, we expectC2E correlation to be weaker than E2E correlation because amplificationin A docs not necessarily imply A is expressed at higher levels, whichin turn is necessary to upregulate B. In this way, each link in apathway provides an expectation about the data; pathways with manyconsistent links may be relevant for further consideration. We testedthese assumptions and found that the NCI pathways contain manyinteractions predictive of the recent TCGA GBM data (The TCGA researchnetwork 2008).

We have developed an approach called PARADIGM (PAthway RecognitionAlgorithm using Data Integration on Genomic Models) to infer theactivities of genetic pathways from integrated patient data.

The PARADIGM method integrates diverse high-throughput genomicsinformation with known signaling pathways to provide patient-specificgenomic inferences on the state of gene activities, complexes, andcellular processes. The core of the method uses a factor graph toleverage inference for combining the various data sources. The use ofsuch inferences in place of, or in conjunction with, the originalhigh-throughput datasets improves our ability to classify samples intoclinically relevant subtypes. Clustering the GBM patients based on thePARADIGM-integrated activities revealed patient subtypes correlated withdifferent survival profiles. In contrast, clustering the samples eitherusing the expression data or the copy-number data did not reveal anysignificant clusters in the dataset.

PARADIGM produces pathway inferences of significantly altered geneactivities in tumor samples from both GBM and breast cancer. Compared toa competing pathway activity inference approach called SPIA, our methodidentifies altered activities in cancer-related pathways with fewerfalse-positives. For computational efficiency, PARADIGM currently usesthe NCI pathways as is.

While it infers hidden quantities using EM, it makes no attempt to infernew interactions not already present in an NCI pathway. One can imagineexpanding the approach to introduce new interactions that increase thelikelihood function. While this problem is intractable in general,heuristics such as structural EM (Friedman (1997) supra) can be used toidentify interactions using computational search strategies.

Rather than searching for novel connections de novo one could speed upthe search significantly by proposing interactions derived fromprotein-protein interaction maps or gene pairs correlated in asignificant number of expression datasets. The power of thepathway-based approach is it may provide clues about the possiblemechanisms underlying the differences in observed survival. InformativeIPAs may be useful for suggesting therapeutic targets or to select themost appropriate patients for clinical trials. For example, the ErbB2amplification is a well-known marker of particular forms of breastcancer that are treatable by the drug trastuzumab.

However, some patients with the ErbB2 amplification have tumors that arerefractory to treatment. Inspection of a CircleMap display couldidentify patients with ErbB2 amplifications but have either inactive orunchanged IPAs as inferred by PARADIGM. Patients harboring the ErbB2amplification but without predicted activity could be considered foralternative treatment.

As more multidimensional datasets become available in the future, itwill be interesting to test whether such pathway inferences providerobust biomarkers that generalize across cohorts.

Subtype and Pathway Specific Responses to Anti-Cancer Compounds inBreast Cancer

More than 800 small molecule inhibitors and biologics are now underdevelopment for treatment of human malignancies (New Medicines DatabaseI PHRMA. newmeds.phrma.org (2010). Many of these agents target molecularfeatures thought to distinguish tumor from normal cells, and range frombroad-specificity conventional therapeutics, including anti-metabolitesand DNA cross-linking agents, such as trastuzumab and lapatinib, thatselectively target molecular events and pathways deregulated in cancersubsets (see for example, Slamon, D. J. et al. Use of chemotherapy plusa monoclonal antibody against HER2 for metastatic breast cancer thatoverexpresses HER2. N Engl J Med 344, 783-792 (2001),Vogel, C. L. et al.Efficacy and safety of trastuzumab as a single agent in first-linetreatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol20, 719-726 (2002); Rusnak, D. W. et al. The effects of the novel,reversible epidermal growth factor receptor/ErbB-2 tyrosine kinaseinhibitor, GW2016, on the growth of human normal and tumor-derived celllines in vitro and in vivo. Mol Cancer Ther 1, 85-94 (2001)). Effects ofchemotherapy and hormonal therapy for early breast cancer on recurrenceand 15-year survival: an overview of the randomised trials. Lancet 365,1687-1717 (2005).

The general trend in drug development today is moving toward targetedagents that show increased efficacy and lower toxicity than conventionalagents (Sawyers, C. Targeted cancer therapy. Nature 432, 294-297(2004)). Some drugs, such as the ERBB2/EGFR inhibitor lapatinib, showhigh target specificity while others, such as the SRC inhibitordasatinib, inhibit a broad range of kinases (Karaman, M. W. et al. Aquantitative analysis of kinase inhibitor selectivity. Nat Biotechnol26, 127-132 (2008)).

There is growing recognition that clinical trials must includepredictors of response and stratify patients entering the trial. Whilemany molecularly targeted therapeutic agents offer obvious molecularfeatures on which to stratify patients, most do not. Moreover, molecularand biological differences between tumors, complex cross-coupling andfeedback regulation of targeted pathways and imprecise targetingspecificity frequently complicate basic mechanistic predictions. Whileresponsive subsets can be identified during the course of molecularmarker based clinical trials, this approach is logistically difficult,expensive, and does not allow experimental compounds to be initiallytested in selected subpopulations most likely to respond. Indeed, themajority of drugs now under development will never be tested in breastcancer, so the probability is high that compounds that are veryeffective only in subpopulations of patients with breast cancer will bemissed. A promising approach is to employ predictors of response derivedfrom preclinical models to stratify patients entering clinical trials,which would reduce development costs and identify those drugs that maybe particularly effective in subsets of patients.

Preclinical testing in panels of cell lines promises to allow early andefficient identification of responsive molecular subtypes as a guide toearly clinical trials. Evidence for the utility of this approach comesfrom studies showing that cell line panels predict (a) lung cancers withEGFR mutations as responsive to gefitinib (Paez, J. G. et al. EGFRmutations in lung cancer: correlation with clinical response togefitinib therapy. Science 304, 1497-1500 (2004)), (b) breast cancerswith HER2/ERBB2 amplification as responsive to trastuzumab and/orlapatinib (Neve, R. M. et al. A collection of breast cancer cell linesfor the study of functionally distinct cancer subtypes. Cancer Cell 10,515-527 (2006); Konecny, G. E. et al. Activity of the dual kinaseinhibitor lapatinib (GW572016) against HER-2-overexpressing andtrastuzumab-treated breast cancer cells. Cancer Res 66, 1630-1639(2006)), and (c) tumors with mutated or amplified BCR-ABL as resistantto imatinib mesylate (Scappini, B. et al. Changes associated with thedevelopment of resistance to imatinib (STI571) in two leukemia celllines expressing p210 Bcr/Abl protein. Cancer 100, 1459-1471 (2004)).The NCI's Discovery Therapeutic Program has pursued this approach onlarge scale, identifying associations between molecular features andresponses to >100,000 compounds in a collection of −60 cancer cell lines(Weinstein, J. N. Spotlight on molecular profiling: “Integromic”analysis of the NCI-60 cancer cell lines. Mol Cancer Ther 5, 2601-2605(2006); Bussey, K. J. et al. Integrating data on DNA copy number withgene expression levels and drug sensitivities in the NCI-60 cell linepanel. Mol Cancer Ther 5, 853-867 (2006)). Although useful for detectingcompounds with diverse responses, the NCI60 panel is arguably of limitedpower in detecting subtype specific responses because of the relativelysparse representation of specific cancer subtypes in the collection. Forexample, the collection carries only 6 breast cancer cell lines, whichis not enough to adequately represent the known heterogeneity. We havetherefore promoted the use of a collection of −50 breast cancer celllines for more statistically robust identification of associationsbetween in vitro therapeutic compound response and molecular subtypesand activated signaling pathways in breast cancer. Here we report theassessment of associations between quantitative growth inhibitionresponses and molecular features defining subtypes and activatedpathways for 77 compounds, including both FDA approved drugs andinvestigational compounds. Approximately half show aberration or subtypespecificity. We also show via integrative analysis of gene expressionand copy number data that some of the observed subtype-associatedresponses can be explained by specific pathway activities.

Integrated Molecular Profiles Reveal Distorted Interleukin Signalling inDcis and Improved Prognostic Power in Invasive Breast Cancer

The accumulation of high throughput molecular profiles of tumors atvarious levels has been a long and costly process worldwide. Combinedanalysis of gene regulation at various levels may point to specificbiological functions and molecular pathways that are deregulated inmultiple epithelial cancers and reveal novel subgroups of patients fortailored therapy and monitoring. We have collected high throughput dataat several molecular levels derived from fresh frozen samples fromprimary tumors, matched blood, and with known micrometastases status,from approximately 110 breast cancer patients (further referred to asthe MicMa dataset). These patients are part of a cohort of over 900breast cancer cases with information about presence of disseminatedtumor cells (DTC), long-term follow-up for recurrence and overallsurvival. The MicMa set has been used in parallel pilot studies of wholegenome mRNA expression (Naume, B. et al., (2007), Presence of bonemarrow micrometastasis is associated with different recurrence riskwithin molecular subtypes of breast cancer, 1: 160-17), arrayCGH(Russnes, H. G. et al., (2010), Genomic architecture characterizes tumorprogression paths and fate in breast cancer patients, 2: 38ra472), DNAmethylation (Ronneberg, J. A. et al., (2011), Methylation profiling witha panel of cancer related genes: association with estrogen receptor,TP53 mutation status and expression subtypes in sporadic breast cancer,5: 61-76), whole genome SNP and SNP-CGH (Van, Loo P. et al., (2010),Allele-specific copy number analysis of tumors, 107: 16910-169154),whole genome miRNA expression analyses (Enerly E, Steinfeld I, Kleivi K,Leivonen S, Aure M R, Russnes H G, Ronneberg J A, Johnsen H, Navon R,Rodland E, Makela R, Naume B, Perath. M, Kallioniemi O, Kristensen V N,Yakhini Z, BOrresen-Dale A. miRNA-mRNA integrated analysis reveals rolesfor miRNAs in primary breast tumors. PLoS ONE 2011; 6(2):e16915). TP53mutation status dependent pathways and high throughput paired endsequencing (Stephens, P. J. et al., (2009), Complex landscapes ofsomatic rearrangement in human breast cancer genomes, 462: 10051010).This is a comprehensive collection of high throughput molecular dataperformed by a single lab on the same set of primary tumors of thebreast.

Below we summarize the findings of these studies, each of which hasattempted to integrate mRNA expression with either DNA copy numbers,deregulation in DNA methylation or miRNA expression. While in the pastwe and others have looked at breast cancer mechanisms on multiplemolecular levels, there has been very sparse attempt to integrate theseviews by modeling mRNA, CNAs, miRNAs, and methylation in a pathwaycontext. In this paper we have analyzed such data from breast cancers inconcert to both detect pathways perturbed and molecular subtypes withdistinct phenotypic characteristics.

In the MicMa dataset discussed here we have identified three majorclusters (and one minor) based on the methylation profiles; one of themajor clusters consisted mainly of tumors of myoepithelial origin andtwo others with tumors of predominantly luminal epithelial origin. Theclusters were different with respect to TP53 mutation and ER, and ErbB2expression status, as well as grade. Pathway analyses identified asignificant association with canonical (curated) pathways includinggenes like EGF, NGFR and TNF, dendritic cell maturation and the NF-KBsignaling pathway. Pyrosequencing of candidate genes on samples fromDCIS's and invasive cancers identified ABCB1, FOXC1, PPP2R2B and PTEN asnovel genes methylated in DCIS. Understanding how these epigeneticchanges are involved in triggering tumor progression is important for abetter understanding of which lesions are “at risk” of becominginvasive.

We have also investigated the relationship between miRNA and mRNAexpression in the MicMa dataset, in terms of their correlation with eachother and with clinical characteristics. We were able to show thatseveral cellular processes, such as proliferation, cell adhesion andimmune response, are strongly associated with certain miRNAs.Statistically significant differential expression of miRNAs was observedbetween molecular intrinsic subtypes, and between samples with differentlevels of proliferation. We validated the role of miRNAs in regulatingproliferation using high-throughput lysate-microarrays on cell lines andpoint to potential drivers of this process (Enerly et al. (2001) supra).

Over 40 KEGG pathways were identified showing differential enrichmentaccording to TP53 mutation status at the p-value cut-off level of 10e-6in this cohort of breast cancer patients. The differential enrichment ofpathways was also observed on the cross-platform dataset consisting of187 breast cancer samples, based on two different microarray platforms.Differentially enriched pathways included several known cancer pathwayssuch as TP53 signaling and cell cycle, signaling pathways includingimmune response and cytokine activation and metabolic pathways includingfatty acid metabolism (Joshi et al, 2011 supra).

Each of the studies described earlier has attempted to derive biologicalinteractions from high throughput molecular data in a pair-wise fashion(CNA/mRNA, miRNA/mRNA, DNAmeth/mRNA, TP53/inRNA). In the present studywe have attempted to focus on the deregulated pathways and develop anintegrated prognostic index taking into account all molecular levelssimultaneously. We applied the Pathway Recognition Algorithm using Dataintegration on Genomic Models (PARADIGM) to elucidate the relativeactivities of various genetic pathways and to evaluate their jointprognostic potential. The clusters and deregulated pathways identifiedby PARADIGM were then validated in another dataset (Chin, S. F. et al.,(2007), Using array-comparative genomic hybridization to definemolecular portraits of primary breast cancers, 26: 1959-1970), and alsostudied in a dataset of premalignant neoplasia such as DCIS, (ductalcarcinoma in situ) (Muggerud, A. A. et al., (2010), Molecular diversityin ductal carcinoma in situ (DCIS) and early invasive breast cancer, 4:357-368).

Frequently Altered Pathways in Ovarian Serous Carcinomas

To identify significantly altered pathways through an integratedanalysis of both copy number and gene expression, we applied therecently developed pathway activity inference method PARADIGM (PM1D:20529912). The computational model incorporates copy number changes,gene expression data, and pathway structures to produce an integratedpathway activity (IPA) for every gene, complex, and genetic processpresent in the pathway database. We use the term “entity” to refer toany molecule in a pathway be it a gene, complex, or small molecule. TheIPA of an entity refers only to the final activity. For a gene, the IPAonly refers to the inferred activity of the active state of the protein,which is inferred from copy number, gene expression, and the signalingof other genes in the pathway. We applied PARADIGM to the ovariansamples and found alterations in many different genes and processespresent in pathways contained in the National Cancer Institutes' PathwayInteraction Database (NCI-PID). We assessed the significance of theinferred alterations using 1000 random simulations in which pathwayswith the same structure were used but arbitrary genes were assigned atdifferent points in the pathway. In other words, one random simulationfor a given pathway kept the set of interactions fixed so that anarbitrary set of genes were connected together with the pathway'sinteractions. The significance of all samples' IPAs was assessed againstthe same null distribution to obtain a significance level for eachentity in each sample. IPAs with a standard deviation of at least 0.1are displayed as a heatmap in FIG. 28.

Table 3 shows the pathways altered by at least three standard deviationswith respect to pennuted samples found by PARADIGM. The FOXM1transcription factor network was altered in the largest number ofsamples among all pathways tested—67% of entities with alteredactivities when averaged across samples. In comparison, pathways withthe next highest level of altered activities in the ovarian cohortincluded PLK1 signaling events (27%), Aurora B signaling (24%), andThromboxane A2 receptor signaling (20%). Thus, among the pathways inNCI-PID, the FOXM1 network harbors significantly more altered activitiesthan other pathways with respect to the ovarian samples.

The FOXM1 transcription factor network was found to be differentiallyaltered in the tumor samples compared to the normal controls in thehighest proportion of the patient samples (FIG. 29). FOXM1 is amultifunctional transcription factor with three known dominant spliceforms, each regulating distinct subsets of genes with a variety of rolesin cell proliferation and DNA repair. The FOXM1c isofonn directlyregulates several targets with known roles in cell proliferationincluding AUKB, PLK1, CDC25, and BIRC5 (PM D:15671063). On the otherhand, the FOXM1b isofonn regulates a completely different subset ofgenes that include the DNA repair genes BRCA2 and XRCC1 (PMID:17101782).CHEK2, which is under indirect control of ATM, directly regulates FOXMIsexpression level.

We asked whether the IPAs of the FOXM1 transcription factor itself weremore highly altered than the IPAs of other transcription factors. Wecompared the FOXM1 level of activity to all of the other 203transcription factors in the NCI-PID. Even compared to othertranscription factors in the NCI set, the FOXM1 transcription factor hadsignificantly higher levels of activity (p<0.0001; K-S test) suggestingfurther that it may be an important signature (FIGS. 30A-30B).

Because FOXM1 is also expressed in many different normal tissues ofepithelial origin, we asked whether the signature identified by PARADIGMwas due to an epithelial signature that would be considered normal inother tissues. To answer this, we downloaded an independent dataset fromGEO (GSE10971) (PMID:18593983) in which fallopian tube epithelium andovarian tumor tissue were microdissected and gene expression wasassayed. We found that the levels of FOXM1 were significantly higher inthe tumor samples compared to the normals, suggesting FOXM1 regulationis indeed elevated in cancerous tissue beyond what is seen in normalepithelial tissue (FIG. 31).

Because the entire cohort for the TCGA ovarian contained samples derivedfrom high-grade serous tumors, we asked whether the FOXM1 signature wasspecific to high-grade serous. We obtained the log expression of FOXM1and several of its targets from the dataset of Etemadmoghadam et al.(2009) (Etemadmoghadam D, deFazio A, Beroukhim R, Mermel C, George J,Getz G, Tothill R, Okamoto A, Raeder M B, AOCS Study Group, Harnett P,Lade S, Akslen L A, Tinker A V, Locandro B, Alsop K, Chiew Y E,Traficante N, Fereday S, Johnson D, Fox S, Sellers W, Urashima M,Salvesen H B, Meyerson M, Bowtell D. Integrated Genome-Wide DNA CopyNumber and Expression Analysis Identifies Distinct Mechanisms of PrimaryChemoresistance in Ovarian Carcinomas. Clinical Cancer Research 2009February; 15(4):1417-1427) in which both low- and high-grade seroustumors had been transcriptionally profiled. This independent dataconfirmed that FOXM1 and several of its targets are significantlyup-regulated in serous ovarian relative to low-grade ovarian cancers(FIG. 32). To determine if the 25 genes in the FOXM1 transcriptionfactor network contained a significant proportion of genes with higherexpression in high-grade disease, we performed a Student's t-test usingthe data from Etemadmoghadam. 723 genes in the genome (5.4%) were foundto be significantly up-regulated in high- versus low-grade cancer at the0.05 significance level (corrected for multiple testing using theBenjamini-Hochberg method). The FOXM1 network was found to have 13 ofits genes (52%) differentially regulated, which is a significantproportion based on the hypergeometric test (P<3.8*10¹²). Thus, highexpression of the FOXM1 network genes does appear to be specificallyassociated with high-grade disease when compared to the expression oftypical genes in the genome.

The role of FOXM1 in many different cancers including breast and lunghas been well documented but its role in ovarian cancer has not beeninvestigated. FOXM1 is a multifunctional transcription factor with threeknown splice forms, each regulating distinct subsets of genes with avariety of roles in cell proliferation and DNA repair. An excerpt ofFOXM1's interaction network relevant to this analysis is shown in FIG.27. The FOXMla isofonn directly regulates several targets with knownroles in cell proliferation including AUKB, PLK1, CDC25, and BIRC5. Incontrast, the FOXMIb isoform regulates a completely different subset ofgenes that include the DNA repair genes BRCA2 and XRCC1. CHEK2, which isunder indirect control of ATM, directly regulates FOXM1's expressionlevel. In addition to increased expression of FOXM1 in most of theovarian patients, a small subset also have increased copy numberamplifications detected by CBS (19% with copy number increases in thetop 5% quantile of all genes in the genome measured). Thus thealternative splicing regulation of FOXM1 may be involved in the controlswitch between DNA repair and cell proliferation. However, there isinsufficient data at this point to support this claim since the exonstructure distinguishing the isoforms and positions of the Exon arrayprobes make it difficult to distinguish individual isoform activities.Future high-throughput sequencing of the mRNA of these samples may helpdetermine the differential levels of the FOXM1 isoforms. The observationthat PARADIGM detected the highest level of altered activity centered onthis transcription factor suggests that FOXM1 resides at a criticalregulatory point in the cell.

Diagnostics

The methods herein described may be used to detect and quantify alteredgene expression, absence/presence versus excess, expression of mRNAs orto monitor mRNA levels during therapeutic intervention. Conditions,diseases or disorders associated with altered expression includeidiopathic pulmonary arterial hypertension, secondary pulmonaryhypertension, a cell proliferative disorder, particularly anaplasticoligodendroglioma, astrocytoma, oligoastrocytoma, glioblastoma,meningioma, ganglioneuroma, neuronal neoplasm, multiple sclerosis,Huntington's disease, breast adenocarcinoma, prostate adenocarcinoma,stomach adenocarcinoma, metastasizing neuroendocrine carcinoma,nonproliferative fibrocystic and proliferative fibrocystic breastdisease, gallbladder cholecystitis and cholelithiasis, osteoarthritis,and rheumatoid arthritis; acquired immunodeficiency syndrome (AIDS),Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, benign prostatichyperplasia, bronchitis, Chediak-Higashi syndrome, cholecystitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, chronicgranulomatous diseases, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polycystic ovary syndrome,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, severe combined immunodeficiency disease (SCID), Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, hemodialysis, extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infection; a disorder ofprolactin production, infertility, including tubal disease, ovulatorydefects, and endometriosis, a disruption of the estrous cycle, adisruption of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, an endometrial or ovarian tumor, a uterinefibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea; adisruption of spermatogenesis, abnormal sperm physiology, benignprostatic hyperplasia, prostatitis, Peyronie's disease, impotence,gynecomastia; actinic keratosis, arteriosclerosis, bursitis, cirrhosis,hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,paroxysmal nocturnal hemoglobinuria, polycythemia vera, primarythrombocythemia, complications of cancer, cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In another aspect, thenucleic acid of the invention.

The methods described herein may be used to detect and quantify alteredgene expression; absence, presence, or excess expression of mRNAs; or tomonitor mRNA levels during therapeutic intervention. Disordersassociated with altered expression include akathesia, Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis, ataxias, bipolardisorder, catatonia, cerebral palsy, cerebrovascular diseaseCreutzfeldt-Jakob disease, dementia, depression, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, muscular dystrophy, neuralgias, neurofibromatosis,neuropathies, Parkinson's disease, Pick's disease, retinitis pigmentosa,schizophrenia, seasonal affective disorder, senile dementia, stroke,Tourette's syndrome and cancers including adenocarcinomas, melanomas,and teratocarcinomas, particularly of the brain.

In order to provide a basis for the diagnosis of a condition, disease ordisorder associated with gene expression, a normal or standardexpression profile is established. This may be accomplished by combininga biological sample taken from normal subjects, either animal or human,with a probe under conditions for hybridization or amplification.Standard hybridization may be quantified by comparing the valuesobtained using normal subjects with values from an experiment in which aknown amount of a substantially purified target sequence is used.Standard values obtained in this manner may be compared with valuesobtained from samples from patients who are symptomatic for a particularcondition, disease, or disorder. Deviation from standard values towardthose associated with a particular condition is used to diagnose thatcondition.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies and in clinical trial orto monitor the treatment of an individual patient. Once the presence ofa condition is established and a treatment protocol is initiated,diagnostic assays may be repeated on a regular basis to determine if thelevel of expression in the patient begins to approximate the level thatis observed in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to months.

Model Systems

Animal models may be used as bioassays where they exhibit a toxicresponse similar to that of humans and where exposure conditions arerelevant to human exposures. Mammals are the most common models, andmost toxicity studies are performed on rodents such as rats or micebecause of low cost, availability, and abundant reference toxicology.Inbred rodent strains provide a convenient model for investigation ofthe physiological consequences of under- or over-expression of genes ofinterest and for the development of methods for diagnosis and treatmentof diseases. A mammal inbred to over-express a particular gene (forexample, secreted in milk) may also serve as a convenient source of theprotein expressed by that gene.

Toxicology

Toxicology is the study of the effects of agents on living systems. Themajority of toxicity studies are performed on rats or mice to helppredict the effects of these agents on human health. Observation ofqualitative and quantitative changes in physiology, behavior,homeostatic processes, and lethality are used to generate a toxicityprofile and to assess the consequences on human health followingexposure to the agent.

Genetic toxicology identifies and analyzes the ability of an agent toproduce genetic mutations. Genotoxic agents usually have common chemicalor physical properties that facilitate interaction with nucleic acidsand are most harmful when chromosomal aberrations are passed along toprogeny. Toxicological studies may identify agents that increase thefrequency of structural or functional abnormalities in progeny ifadministered to either parent before conception, to the mother duringpregnancy, or to the developing organism. Mice and rats are mostfrequently used in these tests because of their short reproductive cyclethat produces the number of organisms needed to satisfy statisticalrequirements.

Acute toxicity tests are based on a single administration of the agentto the subject to determine the symptomology or lethality of the agent.Three experiments are conducted: (a) an initial dose-range-findingexperiment, (b) an experiment to narrow the range of effective doses,and (c) a final experiment for establishing the dose-response curve.

Prolonged toxicity tests are based on the repeated administration of theagent. Rats and dog are commonly used in these studies to provide datafrom species in different families. With the exception ofcarcinogenesis, there is considerable evidence that daily administrationof an agent at high-dose concentrations for periods of three to fourmonths will reveal most forms of toxicity in adult animals.

Chronic toxicity tests, with a duration of a year or more, are used todemonstrate either the absence of toxicity or the carcinogenic potentialof an agent. When studies are conducted on rats, a minimum of three testgroups plus one control group are used, and animals are examined andmonitored at the outset and at intervals throughout the experiment.

Transgenic Animal Models

Transgenic rodents which over-express or under-express a gene ofinterest may be inbred and used to model human diseases or to testtherapeutic or toxic agents. (See U.S. Pat. Nos. 4,736,866; 5,175,383;and 5,767,337; incorporated herein by reference.) In some cases, theintroduced gene may be activated at a specific time in a specific tissuetype during fetal development or postnatally. Expression of thetransgene is monitored by analysis of phenotype or tissue-specific mRNAexpression in transgenic animals before, during, and after challengewith experimental drug therapies.

Embryonic Stem Cells

Embryonic stem cells (ES) isolated from rodent embryos retain thepotential to form an embryo. When ES cells are placed inside a carrierembryo, they resume normal development and contribute to all tissues ofthe live-born animal. ES cells are the preferred cells used in thecreation of experimental knockout and knockin rodent strains. Mouse EScells, such as the mouse 129/SvJ cell line, are derived from the earlymouse embryo and are grown under culture conditions well known in theart. Vectors for knockout strains contain a disease gene candidatemodified to include a marker gene that disrupts transcription and/ortranslation in vivo. The vector is introduced into ES cells bytransformation methods such as electroporation, liposome delivery,microinjection, and the like which are well known in the art. Theendogenous rodent gene is replaced by the disrupted disease gene throughhomologous recombination and integration during cell division.Transformed ES cells are identified, and preferably microinjected intomouse cell blastocysts such as those from the C57BL/6 mouse strain. Theblastocysts are surgically transferred to pseudopregnant dams and theresulting chimeric progeny are genotyped and bred to produceheterozygous or homozygous strains.

ES cells are also used to study the differentiation of various celltypes and tissues in vitro, such as neural cells, hematopoieticlineages, and cardiomyocytes (Bain et al. (1995) Dev. Biol. 168:342-357; Wiles and Keller (1991) Development 111: 259-267; and Klug etal. (1996) J. Clin. Invest. 98: 216-224). Recent developmentsdemonstrate that ES cells derived from human blastocysts may also bemanipulated in vitro to differentiate into eight separate cell lineages,including endoderm, mesoderm, and ectodermnal cell types (Thomson (1998)Science 282: 1145-1147).

Knockout Analysis

In gene knockout analysis, a region of a human disease gene candidate isenzymatically modified to include a non-mammalian gene such as theneomycin phosphotransferase gene (neo; see, for example, Capecchi (1989)Science 244: 1288-1292). The inserted coding sequence disruptstranscription and translation of the targeted gene and preventsbiochemical synthesis of the disease candidate protein. The modifiedgene is transformed into cultured embryonic stem cells (describedabove), the transformed cells are injected into rodent blastulae, andthe blastulae are implanted into pseudopregnant dams. Transgenic progenyare crossbred to obtain homozygous inbred lines.

Knockin Analysis

Totipotent ES cells, present in the early stages of embryonicdevelopment, can be used to create knockin humanized animals (pigs) ortransgenic animal models (mice or rats) of human diseases. With knockintechnology, a region of a human gene is injected into animal ES cells,and the human sequence integrates into the animal cell genome byrecombination. Totipotent ES cells that contain the integrated humangene are handled as described above. Inbred animals are studied andtreated to obtain information on the analogous human condition. Thesemethods have been used to model several human diseases. (See, forexample, Lee et al. (1998) Proc. Natl. Acad. Sci. 95: 11371-11376;Baudoin et al. (1998) Genes Dev. 12: 1202-1216; and Zhuang et al. (1998)Mol. Cell Biol. 18: 3340-3349).

Non-Human Primate Model

The field of animal testing deals with data and methodology from basicsciences such as physiology, genetics, chemistry, pharmacology andstatistics. These data are paramount in evaluating the effects oftherapeutic agents on non-human primates as they can be related to humanhealth. Monkeys are used as human surrogates in vaccine and drugevaluations, and their responses are relevant to human exposures undersimilar conditions. Cynomolgus monkeys (Macaca fascicularis, Macacamulata) and common marmosets (Callithrix jacchus) are the most commonnon-human primates (NHPs) used in these investigations. Since great costis associated with developing and maintaining a colony of NHPs, earlyresearch and toxicological studies are usually carried out in rodentmodels. In studies using behavioral measures such as drug addiction,NHPs are the first choice test animal. In addition, NHPs and individualhumans exhibit differential sensitivities to many drugs and toxins andcan be classified as “extensive metabolizers” and “poor metabolizers” ofthese agents.

Exemplary Uses of the Invention

Personalized medicine promises to deliver specific treatment(s) to thosepatients mostly likely to benefit. We have shown that approximately halfof therapeutic compounds are preferentially effective in one or more ofthe clinically-relevant transcriptional or genomic breast cancersubtypes. These findings support the importance of definingresponse-related molecular subtypes in breast cancer treatment. We alsoshow that pathway integration of the transcriptional and genomic data onthe cell lines reveals subnetworks that provide mechanistic explanationsfor the observed subtype specific responses. Comparative analysis ofsubnet activities between cell lines and tumors shows that the majorityof subtype-specific subnetworks are conserved between cell lines andtumors. These analyses support the idea that preclinical screening ofexperimental compounds in a well-characterized cell line panel canidentify candidate response-associated molecular signatures that can beused for sensitivity enrichment in early-phase clinical trials. Wesuggest that this in vitro assessment approach will increase thelikelihood that responsive tumor subtypes will be identified before acompound's clinical development begins, thereby reducing cost,increasing the probability of eventual FDA approval and possiblyavoiding toxicity associated with treating patients unlikely to respond.In this study we have assessed only molecular signatures that definetranscriptional subtypes and selected recurrent genome CNAs. Weanticipate that the power and precision of this approach will increaseas additional molecular features such as genetic mutation, methylationand alternative splicing, are included in the analysis. Likewise,increasing the size of the cell line panel will increase the power toassess less common molecular patterns within the panel and increase theprobability of representing a more complete range of the diversity thatexists in human breast cancers.

Breast cancer development is characterized by significant increases inthe presence of both innate and adaptive immune cells, with B cells, Tcells, and macrophages representing the most abundant leukocytes presentin neoplastic stroma (DeNardo D G, Coussens L M. Inflammation and breastcancer. Balancing immune response: crosstalk between adaptive and innateimmune cells during breast cancer progression. Breast Cancer Res. 2007;9(4):212). High immunoglobulin (Ig) levels in tumor stoma (andserum),and increased presence of extra follicular B cells, T regulatory cells,and high ratios of CD4/CD8 or TH2/TH1 T lymphocytes in primary tumors orin lymph nodes have been shown to correlate with tumor grade, stage, andoverall patient survival (Bates, G. J. et al., (2006), Quantification ofregulatory T cells enables the identification of high-risk breast cancerpatients and those at risk of late relapse, 24: 5373-5380); Someleukocytes exhibit antitumor activity, including cytotoxic T lymphocytes(CTLs) and natural killer (NK) cells (34 Dunn, G. P., Koebel, C. M., andSchreiber, R. D., (2006), Interferons, immunity and cancerimmunoediting, 6: 836-848), other leukocytes, such as mast cells,Bcells, dendritic cells, granulocytes, and macrophages, exhibit morebipolar roles, through their capacity to either hamper or potentiatetumor progression (35 de Visser, K. E. and Coussens, L. M., (2006), Theinflammatory tumor microenvironment and its impact on cancerdevelopment, 13: 118-137). The most prominent finding in these studieswas the identification of the perturbation in the immune response (TCR)and interleukin signaling, IL4, IL6, IL12 and IL23 signaling leading toclassification of subclasses with prognostic value. We provide hereevidence that these events are mirrored in high throughput moleculardata and interfere strongly with molecular sub-classification of breasttumors.

This disclosure also provides the first large scale integrative view ofthe aberrations in HGS-OvCa. Overall, the mutational spectrum wassurprisingly simple. Mutations in TP53 predominated, occurring in atleast 96% of HGS-OvCa while BRCA1/2 were mutated in 22% of tumors due toa combination of germline and somatic mutations. Seven othersignificantly mutated genes were identified, but only in 2-6% ofHGS-OvCa. In contrast, HGS-OvCa demonstrates a remarkable degree ofgenomic disarray. The frequent SCNAs are in striking contrast toprevious TCGA findings with glioblastoma46 where there were morerecurrently mutated genes with far fewer chromosome arm-level or focalSCNAs (FIG. 37A). A high prevalence of mutations and promotermethylation in putative DNA repair genes including HR components mayexplain the high prevalence of SCNAs. The mutation spectrum marksHGS-OvCa as completely distinct from other OvCa histological subtypes.For example, clear-cell OvCa have few TP53 mutations but have recurrentARID1A and PIK3CA47-49 mutations: endometrioid OvCa have frequentCTINBI, ARID1A, and PIK3CA mutations and a lower rate of TP5348,49 whilemucinous OvCa have prevalent KRAS mutations50. These differences betweenovarian cancer subtypes likely reflect a combination of etiologic andlineage effects, and represent an opportunity to improve ovarian canceroutcomes through subtype-stratified care.

Identification of new therapeutic approaches is a central goal of theTCGA. The −50% of HGS-OvCa with HR defects may benefit from PARPinhibitors. Beyond this, the commonly deregulated pathways, RB,RAS/PI3K, FOXM1, and NOTCH, provide opportunities for therapeuticattack. Finally, inhibitors already exist for 22 genes in regions ofrecurrent amplification (see Examples XIII et seq.), warrantingassessment in HGS-OvCa where the target genes are amplified. Overall,these discoveries set the stage for approaches to treatment of HGS-OvCain which aberrant genes or networks are detected and targeted withtherapies selected to be effective against these specific aberrations.

In additional embodiments, the polynucleotide nucleic acids may be usedin any molecular biology techniques that have yet to be developed,provided the new techniques rely on properties of nucleic acid moleculesthat are currently known, including, but not limited to, such propertiesas the triplet genetic code and specific base pair interactions.

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

EXAMPLES Example I: Data Sources

Breast cancer copy number data from Chin (2007 supra) was obtained fromNCBI Gene Expression Omnibus (GEO) under accessions GPL5737 withassociated array platform annotation from GSE8757.

Probe annotations were converted to BED15 format for display in the UCSCCancer Genomics Browser (Zhu:2009, supra) and subsequent analysis. Arraydata were mapped to probe annotations via probe ID. Matched expressiondata from Naderi (2007, supra) was obtained from MIAM1Express at EBIusing accession number E-UCon-1. Platform annotation information forHuman 1A (V2) was obtained from the Agilent website. Expression data wasprobe-level median-normalized and mapped via probe ID to HUGO genenames.

All data was non-parametrically normalized using a ranking procedureincluding all sample-probe values and each gene-sample pair was given asigned p-value based on the rank. A maximal p-value of 0.05 was used todetermine gene-samples pairs that were significantly altered.

The glioblastoma data from TCGA was obtained from the TCGA Data Portalproviding gene expression for 230 patient samples and 10 adjacent normaltissues on the Affymetrix U133A platform. The probes for the patientsamples were normalized to the normal tissue by subtracting the mediannormal value of each probe. In addition, CBS segmented (Olshen:2004supra p1618) copy number data for the same set of patients wereobtained. Both datasets were non-parametrically normalized using thesame procedure as the breast cancer data.

Example II: Pathway Compendium

We collected the set of curated pathways available from the NationalCancer Institute Pathway Interaction Database (NCI PID) (Schaefer:2009supra). Each pathway represents a set of interactions logically groupedtogether around high-level biomolecular processes describing intrinsicand extrinsic sub-cellular-, cellular-, tissue-, or organism-levelevents and phenotypes. BioPAX level 2 formatted pathways weredownloaded. All entities and interactions were extracted with SPARQLqueries using the Rasqal RDF engine.

We extracted five different types of biological entities (entities)including three physical entities (protein-coding genes, smallmolecules, and complexes), gene families, and abstract processes. A genefamily was created whenever the cross-reference for a BioPAX proteinlisted proteins from distinct genes. Gene families represent collectionsof genes in which any single gene is sufficient to perform a specificfunction. For example, homologs with redundant roles and genes found tofunctionally compensate for one another are combined into families.

The extraction produced a list of every entity and interaction used inthe pathway with annotations describing their different types. We alsoextracted abstract processes, such as “apoptosis,” that refer to generalprocesses that can be found in the NCI collection. For example, pathwaysdetailing the interactions involving the p53 tumor suppressor geneinclude links into apoptosis and senescence that can be leveraged asfeatures for machine-learning classification.

As expected, C2E correlations were moderate, but had a strikingenrichment for positive correlations among activating interactions thanexpected by chance (FIG. 3). E2E correlations were even stronger andsimilarly enriched. Thus, even in this example of a cancer that haseluded characterization, a significant subset of pathway interactionsconnect genomic alterations to modulations in gene expression,supporting the idea that a pathway-level approach is worth pursuing.

Example III: Modeling and Predicting Biological Pathways

We first converted each NCI pathway into a distinct probabilistic model.A toy example of a small fragment of the p53 apoptosis pathway is shownin FIGS. 2A-2C. A pathway diagram from NCI was converted into a factorgraph that includes both hidden and observed states. The factor graphintegrates observations on gene- and biological process-related stateinformation with a structure describing known interactions among theentities.

To represent a biological pathway with a factor graph, we use variablesto describe the states of entities in a cell, such as a particular mRNAor complex, and use factors to represent the interactions andinformation flow between these entities. These variables represent the\textit{differential state of each entity in comparison to a “control”or normal level rather than the direct concentrations of the molecularentities. This representation allows us to model many high-throughputdatasets, such as gene expression detected with DNA microarrays, thatoften either directly measure the differential state of a gene orconvert direct measurements to measurements relative to matchedcontrols. It also allows for many types of regulatory relationshipsamong genes. For example, the interaction describing MDM2 mediatingubiquitin-dependent degradation of p53 can be modeled as activated MDM2inhibiting p53's protein level.

The factor graph encodes the state of a cell using a random variable foreach entity X=x₁, x₁ . . . , x_(n),} and a set of m non-negativefunctions, or factors, that constrain the entities to take onbiologically meaningful values as functions of one another. The j-thfactor I defines a probability distribution over a subset of entitiesX_(j)⊂X.

The entire graph of entities and factors encodes the joint probabilitydistribution over all of the entities as:

$\begin{matrix}{{P(X)} = {\frac{1}{Z}{\prod\;{\begin{matrix}m \\{j = 1}\end{matrix}{\Phi_{j}\left( X_{j} \right)}}}}} & (1)\end{matrix}$where Z=Π_(j)Σ_(s xj)Φ_(j)(S) is a normalization constant and S Xdenotesthat S is a ‘setting’ of the variables in X.

Each entity can take on one of three states corresponding to activated,nominal, or deactivated relative to a control level (for example, asmeasured in normal tissue) and encoded as 1, 0, or −1 respectively. Thestates may be interpreted differently depending on the type of entity(for example, gene, protein, etc). For example, an activated mRNA entityrepresents overexpression, while an activated genomic copy entityrepresents more than two copies are present in the genome.

FIGS. 2A-2C show the conceptual model of the factor graph for a singleprotein-coding gene. For each protein-coding gene G in the pathway,entities are introduced to represent the copy number of the genome(G_(DNA)), mRNA expression (G_(mRNA)), protein level (G_(protein)), andprotein activity (G_(protein)) (ovals labeled “DNA”, “mRNA”, “protein”,and “active” in FIGS. 2A-2C). For every compound, protein complex, genefamily, and abstract process in the pathway, we include a singlevariable with molecular type “active.”

While the example in FIGS. 2A-2C show only one process (“Apoptosis”), inreality many pathways have multiple such processes that representeverything from outputs (for example, “Apoptosis” and “Senescence”) toinputs (for example, “DNA damage”) of gene activity.

In order to simplify the construction of factors, we first convert thepathway into a directed graph, with each edge in the graph labeled witheither positive or negative influence. First, for every protein codinggene G, we add edges with a label “positive” from G_(DNA) to G_(mRNA)from G_(mRNA) to G_(mRNA) tO G_(protein) and from G_(protein) toG_(protein) to reflect the expression of the gene from its number ofcopies to the presence of an activated form of its protein product.Every interaction in the pathway is converted to a single edge in thedirected graph.

Using this directed graph, we then construct a list of factors tospecify the factor graph. For every variable x_(i), we add a singlefactor Φ(X_(i)), where X_(i)={x_(i)}∪{Parents}(x_(i))} and Parents(x_(i)) refers to all the parents of x_(i) in the directed graph. Thevalue of the factor for a setting of all values is dependent on whetherx_(i) is in agreement with its expected value due to the settings ofParents (x_(i)).

For this study, the expected value was set to the majority vote of theparent variables. If a parent is connected by a positive edge itcontributes a vote of +1 times its own state to the value of the factor.Conversely, if the parent is connected by a negative edge, then thevariable votes −1 times its own state. The variables connected to x_(i)by an edge labeled “minimum” get a single vote, and that vote's value isthe minimum value of these variables, creating an AND-like connection.Similarly the variables connected to x_(i) by an edge labeled “maximum”get a single vote, and that vote's value is the maximum value of thesevariables, creating an OR-like connection. Votes of zero are treated asabstained votes. If there are no votes the expected state is zero.Otherwise, the majority vote is the expected state, and a tie between 1and −1 results in an expected state of −1 to give more importance torepressors and deletions. Given this definition of expected state,Φ_(i)(x_(i), Parents(x_(i))) is specified as:

${\Phi_{i}\left( {x_{i},{{Parents}\left( x_{i} \right)}} \right)} = \left\{ \begin{matrix}{1 - {ɛx_{i}\mspace{14mu}{is}\mspace{14mu}{the}\mspace{14mu}{expecteed}\mspace{14mu}{state}\mspace{14mu}{from}\mspace{14mu}{Parents}\;\left( x_{i} \right)}} \\{\frac{ɛ}{2}\mspace{14mu}{otherwise}}\end{matrix} \right.$

For the results shown here, E was set to 0.001, but orders of magnitudedifferences in the choice of epsilon did not significantly affectresults. Finally, we add observation variables and factors to the factorgraph to complete the integration of pathway and multi-dimensionalfunctional genomics data (FIGS. 2A-2C). Each discretized functionalgenomics dataset is associated with one of the molecular types of aprotein-coding gene.

Array CGH/SNP estimates of copy number alteration are associated withthe ‘genome’ type. Gene expression data is associated with the ‘mRNA’type. Though not presented in the results here, future expansion willinclude DNA methylation data with the ‘mRNA’ type, and proteomics andgene-resequencing data with the ‘protein’ and ‘active’ types. Eachobservation variable is also ternary valued. The factors associated witheach observed type of data are shared across all entities and learnedfrom the data, as described next.

Example IV: Inference and Parameter Estimation

Let the set of assignments D={x₁=s₁, x₂=s₂, x₂, . . . , X_(k)=S_(k),}represent a complete set of data for a patient on the observed variablesindexed 1 through k. Let {S_(D) X} represent the set of all possibleassignments of a set of variables X that are consistent with theassignments in D; i.e. any observed variables x₁ are fixed to theirassignments in D while hidden variables can vary.

Given patient data, we would like to estimate whether a particularhidden entity x, is likely to be in state a, for example, how likelyTP53's protein activity is −1 (inactivated) or ‘Apoptosis’ is +1(activated). To do this, we must compute the prior probability of theevent prior to observing patient's data. If A_(i)(a) represents thesingleton assignment set {x₁=a} and Φ is the fully specified factorgraph, this prior probability is:

$\begin{matrix}{{{P\left( {x_{i} = {a❘\Phi}} \right)} = {\frac{1}{Z}{\prod\;{\begin{matrix}m \\{j = 1}\end{matrix}{\sum_{S \Subset_{A_{i}{(a)}}X_{j}}\;{\Phi_{j}(S)}}}}}},} & (2)\end{matrix}$where Z is the normalization constant introduced in Equation (1).Similarly, the probability of x_(i) is in state a along with all of theobservations for the patient is:

$\begin{matrix}{{P\left( {{x_{i} = a},{D❘\Phi}} \right)} = {\frac{1}{Z}{\prod\;{\begin{matrix}m \\{j = 1}\end{matrix}{\sum\limits_{S \Subset_{{A_{i}{(a)}}\bigcup D}X_{j}}{{\Phi_{j}(S)}.}}}}}} & (3)\end{matrix}$

We used the junction tree inference algorithm with HUGIN updates for themajority of pathways. For pathways that take longer than 3 seconds ofinference per patient, we use Belief Propagation with sequentialupdates, a convergence tolerance of 10⁻⁹, and a maximum of 10,000iterations. All inference was performed in the real domain, as opposedto the log domain, and was performed with libDAl (Mooij:2009 supra).

To learn the parameters of the observation factors we use theExpectation-Maximization (EM) algorithm (Dempster (1977) supra).Briefly, EM learns parameters in models with hidden variables byiterating between inferring the probabilities of hidden variables andchanging parameters to maximize likelihood given the probabilities ofhidden variables. We wrote and contributed code to libDAl to perform EM.For each pathway, we created a factor graph for each patient, appliedthe patient's data, and ran EM until the likelihood changed less than0.1%. We averaged the parameters learned from each pathway, and thenused these parameters to calculate final posterior beliefs for eachvariable.

After inference, we output an integrated pathway activity for eachvariable that has an “active” molecular type. We computed alog-likelihood ratio using quantities from equations 2 and 3 thatreflects the degree to which a patient's data increases our belief thatentity i's activity is up or down:

$\begin{matrix}\begin{matrix}{{L\left( {i,a} \right)} = {{\log\left( \frac{P\left( {{D_{1}x_{i}} = {a❘\Phi}} \right.}{P\left( {{{D_{1}x_{i}} \neq a}❘\Phi} \right)} \right)} - {\log\left( \frac{P\left( {x_{i} = {a❘\Phi}} \right)}{P\left( {{x_{i} \neq a}❘\Phi} \right)} \right)}}} \\{= {{\log\left( \frac{P\left( {{D❘x_{i}} = {a_{1}\Phi}} \right)}{P\left( {D❘{x_{i} \neq {a_{1}\Phi}}} \right)} \right)} \cdot}}\end{matrix} & (4)\end{matrix}$

We then computed a single integrated pathway activity (IPA) for gene ibased on the log-likelihood ratio as:

$\begin{matrix}{{{IPA}(i)} = \left\{ \begin{matrix}{L\left( {i,1} \right)} & {{L\left( {i,1} \right)} > {{L\left( {i,{- 1}} \right)}\mspace{14mu}\text{and}\mspace{14mu}{L\left( {i,1} \right)}} > {L\left( {i,0} \right)}} \\{- {L\left( {i,{- 1}} \right)}} & {{L\left( {i,{- 1}} \right)} > {{L\left( {i,1} \right)}\mspace{14mu}\text{and}\mspace{14mu}{L\left( {i,{- 1}} \right)}} > {L\left( {i,0} \right)}} \\0 & \text{otherwise}\end{matrix} \right.} & (5)\end{matrix}$

Intuitively, the IPA score reflects a signed analog of thelog-likelihood ratio, L.

If the gene is more likely to be activated, the IPA is set to L.Alternatively, if the gene is more likely to be inactivated, the IPA isset to the negative of the log likelihood ratio. If the gene is mostlikely unchanged, the IPA is set to zero. Each pathway is analyzedindependently of other pathways. Therefore, a gene can be associatedwith multiple inferences, one for each pathway in which it appears.Differing inferences for the same gene can be viewed as alternativeinterpretations of the data as a function of the gene's pathway context.

Example V: Significance Assessment

We assess the significance of IPA scores by two different permutationsof the data. For the “within” permutation, a permuted data sample iscreated by choosing a new tuple of data (i.e. matched gene expressionand gene copy number) first by choosing a random real sample, and thenchoosing a random gene from within the same pathway, until tuples havebeen chosen for each gene in the pathway. For the “any” permutation, theprocedure is the same, but the random gene selection step could choose agene from anywhere in the genome. For both permutation types, 1,000permuted samples are created, and the perturbation scores for eachpermuted sample is calculated. The distribution of perturbation scoresfrom permuted samples is used as a null distribution to estimate thesignificance of true samples.

Example VI: Signaling Pathway Impact Analysis (SPIA)

Signaling Pathway Impact Analysis (SPIA) from Tarca (2009, supra) wasimplemented in C to reduce runtime and to be compatible with ouranalysis environment. We also added the ability to offer more verboseoutput so that we could directly compare SPIA and PARADIGM outputs. Ourversion of SPIA can output the accumulated perturbation and theperturbation factor for each entity in the pathway. This code isavailable upon request.

Example VII: Decoy Pathways

A set of decoy pathways was created for each cancer dataset. Each NCIpathway was used to create a decoy pathway which consisted of the samestructure but where every gene in the pathway was substituted for arandom gene in RefGene. All complexes and abstract processes were keptthe same and the significance analysis for both PARADIGM and SPIA wasrun on the set of pathways containing both real and decoy pathways. Thepathways were ranked within each method and the fraction of real versustotal pathways was computed and visualized.

Example VIII: Clustering and Kaplan-Meier Analysis

Uncentered correlation hierarchical clustering with centroid linkage wasperformed on the glioblastoma data using the methods from Eisen (1998supra p1621). Only IPAs with a signal of at least 0.25 across 75 patientsamples were used in the clustering. By visual inspection, four obviousclusters appeared and were used in the Kaplan-Meier analysis. TheKaplan-Meier curves were computed using R and p-values were obtained viathe log-rank statistic.

Example IX: Validation of PARADIGM

To assess the quality of the EM training procedure, we compared theconvergence of EM using the actual patient data relative to a nulldataset in which tuples of gene expression and copy number (E,C) werepermuted across the genes and patients. As expected, PARADIGM convergedmuch more quickly on the true dataset relative to the null. As anexample, we plotted the IPAs for the gene AKT I as a function of the EMiteration (FIG. 4). One can see that the activities quickly converge inthe first couple of iterations. EM quickly converged to an activatedlevel when trained with the actual patient data whereas it converged toan unchanged activity when given random data. The convergence suggeststhe pathway structures and inference are able to successfully identifypatterns of activity in the integrated patient data.

We next ran PARADIGM on both breast cancer and GBM cohorts. We developeda statistical simulation procedure to determine which IPAs aresignificantly different than what would be expected from a negativedistribution. We constructed the negative distribution by permutingacross all of the patients and across the genes in the pathway.Empirically, we found that permuting only among genes in the pathway wasnecessary to help correct for the fact that each gene has a differenttopological context determined by the network. In the breast cancerdataset, 56,172 IPAs (7% of the total) were found to be significantlyhigher or lower than the matched negative controls. On average, NCIpathways had 497 significant entities per patient and 103 out of 127pathways had at least one entity altered in 20% or more of the patients.In the GBM dataset, 141,682 IPAs (9% of the total) were found to besignificantly higher or lower than the matched negative controls. Onaverage, NCI pathways had 616 significant entities per patient and 110out of 127 pathways had at least one entity altered in 20% or more ofthe patients.

As another control, we asked whether the integrated activities could beobtained from arbitrary genes connected in the same way as the genes inthe NCI pathways. To do this, we estimated the false discovery rate andcompared it to SPIA (Tarca: 2009 supra). Because many genetic networkshave been found to be implicated in cancer, we chose to use simulated“decoy” pathways as a set of negative controls. For each NCI pathway, weconstructed a decoy pathway by connecting random genes in the genometogether using the same network structure as the NCI pathway.

We then ran PARADIGM and SPIA to derive IPAs for both the NCI and decoypathways. For PARADIGM, we ranked each pathway by the number of IPAsfound to be significant across the patients after normalizing by thepathway size. For SPIA, pathways were ranked according to their computedimpact factor. We found that PARADIGM excludes more decoy pathways fromthe top-most activated pathways compared to SP1A (FIG. 5). For example,in breast cancer, PARADIGM ranks 1 decoy in the top 10, 2 in the top 30,and 4 in the top 50. In comparison, SPIA ranks 3 decoys in the top 10,12 in the top 30, and 22 in the top 50. The overall distribution ofranks for NCI IPAs are higher in PARADIGM than in SPIA, observed byplotting the cumulative distribution of the ranks (P 4 0.009, K-S test).

Example X: Top PARADIGM Pathways in Breast Cancer and GBM

We sorted the NCI pathways according to their average number ofsignificant IPAs per entity detected by our permutation analysis andcalculated the top 15 in breast cancer (Table 1) and GBM (Table 2)

Several pathways among the top fifteen have been previously implicatedin their respective cancers. In breast cancer, both SPIA and PARADIGMwere able to detect the estrogen- and ErbB2-related pathways. In arecent major meta-analysis study (Wirapati P, Sotiriou C, Kunkel S,Farmer P, Pradervand S, Haibe-Kains B, Desmedt C, Ignatiadis M, SengstagT, Schutz F, Goldstein D R, Piccart M, Delorenzi M. Meta-analysis ofgene expression profiles in breast cancer: toward a unifiedunderstanding of breast cancer subtyping and prognosis signatures.Breast Cancer Res. 2008; 10(4):R65.), Wirapeti et al. found thatestrogen receptor and ErbB2 status were two of only three key prognosticsignatures in breast cancer. PARADIGM was also able to identify anAKT1-related PI3K signaling pathway as the top-most pathway withsignificant IPAs in several samples (see FIG. 6).

TABLE 1 Top PARADIGM pathways in breast cancer Rank Name Avg. SPIA? 1Class I P13K signaling events mediated by Akt 20.7 No 2 Nectin adhesionpathway 14.1 No 3 Insulin-mediated glucose transport 13.8 No 4ErbB2IErbB3 signaling events 12.1 Yes 5 p75(NTR)-mediated signaling 11.5No 6 HIF-1-alpha transcription factor network 10.7 No 7 Signaling eventsmediated by PTP1B 10.7 No 8 Plasma membrane estrogen inceptor signaling10.6 Yes 9 TCR signaling in naive CD8+ T cells 10.6 No 10 Angiopoietinreceptor Tie2-mediated signaling 10.1 No 11 Class 113 P13K non-lipidkinase events 10.0 No 13 Osteoponlin-mediated events • 9.9 Yes 12IL4-mediated signaling events 9.8 No 14 Enclothel ins 9.8 No 15Neurotrophic factor-mediated Trk signaling 9.7 No aAverage number ofsamples in which significant activity was detected per entity. ^(b)Ycsif the pathway was also ranked in SPINS top 15; No otherwise.

TABLE 2 Ibp PARADIGM pathways in GBM Rank Name Avg. SPIA? 1 Signaling byRet tyrosine kinase 46.0 No 2 Signaling events activated by HepatocyteGFR 43.7 No 3 Endothelins 42.5 Yes 4 Arf6 downstream pathway 42.3 No 5Signaling events mediated by HDAC Class III 36.3 No 6 FOXM1transcription factor network 35.9 Yes 7 IL6-mediated signaling events33.2 No 8 FoxO family signaling 31.3 No 9 IPA receptor mediated events30.7 Yes 10 ErbB2JErbB3 signaling events 30.1 No 11 Signaling mediatedby p38-alpha and p38-beta 28.1 No 12 HIF-1-alpha transcription factornetwork 27.6 Yes 13 Non-genotropic Androgen signaling 27.3 No 14 p38MAPK signaling pathway 27.2 No 15 IL2 signnling events mediated by P13K26.9 No Average number of samples in which significant activity wasdetected per entity. Yes if the pathway was also ranked in SPIA's top15; No otherwise.

The anti-apoptotic AKT1 serine-threonine kinase is known to be involvedin breast cancer and interacts with the ERBB2 pathway (Ju X, Katiyar S,Wang C, Liu M, Jiao X, Li S. Zhou J, Turner J, Lisanti M P, Russell R G,Mueller S C, Ojeifo J, Chen W S, Hay N, Pestell R G. Aktl governs breastcancer progression in vivo. Proc. Natl. Acad. Sci. U.S.A. 2007 May;104(18):7438-7443). In GBM, both FOXM1 and HIF-1-alpha transcriptionfactor networks have been studied extensively and shown to beoverexpressed in high-grade glioblastomas versus lower-grade gliomas(Liu M, Dai B, Kang S, Ban K, Huang F, Lang F F, Aldape K D, Xie T,Pelloski C E, Xie K, Sawaya R, Huang S. FOXM1B is overexpressed in humanglioblastomas and critically regulates the tumorigenicity of gliomacells. Cancer Res. 2006 April; 66(7):3593-3602; Semenza G L. HIF-1 andhuman disease: one highly involved factor. Genes Dev. 2000 August;14(16):1983-1991).

Example XI: Visualization of the Datasets

To visualize the results of PARADIGM inference, we developed a“CircleMap” visualization to display multiple datasets centered aroundeach gene in a pathway (FIG. 7). In this display, each gene isassociated with all of its data across the cohort by plotting concentricrings around the gene, where each ring corresponds to a single type ofmeasurement or computational inference. Each tick in the ringcorresponds to a single patient sample while the color corresponds toactivated (red), deactivated (blue), or unchanged (white) levels ofactivity. We plotted CircleMaps for a subset of the ErbB2 pathway andincluded ER status, IPAs, expression, and copy number data from thebreast cancer cohort.

Gene expression data has been used successfully to define molecularsubtypes for various cancers. Cancer subtypes have been found thatcorrelate with different clinical outcomes such as drug sensitivity andoverall survival. We asked whether we could identify informativesubtypes for GBM using PARADIGM IPAs rather than the raw expressiondata. The advantage of using IPAs is they provide a summarization ofcopy number, expression, and known interactions among the genes and maytherefore provide more robust signatures for elucidating meaningfulpatient subgroups. We first determined all IPAs that were at leastmoderately recurrently activated across the GBM samples and found that1,755 entities had IPAs of 0.25 in at least 75 of the 229 samples. Wecollected all of the IPAs for these entities in an activity matrix. Thesamples and entities were then clustered using hierarchical clusteringwith uncentcred Pearson correlation and centroid linkage (FIG. 8).

Visual inspection revealed four obvious subtypes based on the IPAs withthe fourth subtype clearly distinct from the first three. The fourthcluster exhibits clear downregulation of REF-I-alpha transcriptionfactor network as well as overexpression of the E2F transcription factornetwork. HIF-1-alpha is a master transcription factor involved inregulation of the response to hypoxic conditions. In contrast, two ofthe first three clusters have elevated EGFR signatures and an inactiveMAP kinase cascade involving the GATA interleukin transcriptionalcascade. Interestingly, mutations and amplifications in EGFR have beenassociated with high grade gliomas as well as glioblastomas (Kuan C T,Wikstrand C J, Bigner D D. EGF mutant receptor vIII as a moleculartarget in cancer therapy. Endocr. Relat. Cancer 2001 June; 8(2):83-96).Amplifications and certain mutations can create a constitutively activeEGFR either through self stimulation of the dimer or throughligand-independent activation. The constitutive activation of EGFR maypromote oncogenesis and progression of solid tumors. Gefitinib, amolecule known to target EGFR, is currently being investigated for itsefficacy in other EGFR-driven cancers. Thus, qualitatively, the clustersappeared to be honing in on biologically meaningful themes that canstratify patients.

To quantify these observations, we asked whether the different GBMsubtypes identified by PARADIGM coincided with different survivalprofiles. We calculated Kaplan-Meier curves for each of the fourclusters by plotting the proportion of patients surviving versus thenumber of months after initial diagnosis. We plotted Kaplan-Meiersurvival curves for each of the four clusters to see if any clusterassociated with a distinct IPA signature was predictive of survivaloutcome (FIG. 9). The fourth cluster is significantly different from theother clusters (P<2.11×10; Cox proportional hazards test). Half of thepatients in the first three clusters survive past 18 months; thesurvival is significantly increased for cluster 4 patients where halfsurvive past 30 months. In addition, over the range of 20 to 40 months,patients in cluster 4 are twice as likely to survive as patients in theother clusters.

Example XII: Kaplan-Meier Survival Plots for the Clusters

The survival analysis revealed that the patients in cluster 4 have asignificantly better survival profile. Cluster 4 was found to have anup-regulation of E2F, which acts with the retinoblastoma tumorsuppressor. Up-regulation of E2F is therefore consistent with an activesuppression of cell cycle progression in the tumor samples from thepatients in cluster 4. In addition, cluster 4 was associated with aninactivity of the HIF-1-alpha transcription factor. The inactivity inthe fourth cluster may be a marker that the tumors are more oxygenated,suggesting that they may be smaller or newer tumors. Thus, PARADIGM IPAsprovide a meaningful set of profiles for delineating subtypes withmarkedly different survival outcomes.

For comparison, we also attempted to cluster the patients using onlyexpression data or CNA data to derive patient subtypes. No obviousgroups were found from clustering using either of these data sources,consistent with the findings in the original TCGA analysis of thisdataset (TCGA:2008) (see FIGS. 14A-14B). This suggests that theinteractions among genes and resulting combinatorial outputs ofindividual gene expression may provide a better predictor of such acomplex phenotype as patient outcome.

Example XIII: Integrated Genomic Analyses of Ovarian Carcinoma: Samplesand Clinical Data

This report covers analysis of 489 clinically annotated stage II-IVHGS-OvCa and corresponding normal DNA. Patients reflected the age atdiagnosis, stage, tumor grade, and surgical outcome of individualsdiagnosed with HGS-OvCa. Clinical data were current as of Aug. 25, 2010.HGS-OvCa specimens were surgically resected before systemic treatmentbut all patients received a platinum agent and 94% received a taxane.The median progression-free and overall survival of the cohort issimilar to previously published trials11,12. Twenty five percent of thepatients remained free of disease and 45% were alive at the time of lastfollow-up, while 31% progressed within 6 months after completingplatinum-based therapy. Median follow up was 30 months (range 0 to 179).Samples for TCGA analysis were selected to have >70% tumor cell nucleiand <20% necrosis.

Coordinated molecular analyses using multiple molecular assays atindependent sites were carried out as listed in Table 4 (Data areavailable at tcga.cancer.gov/dataportal) in two tiers. Tier one datasetsare openly available, while tier two datasets include clinical orgenomic information that could identify an individual hence requirequalification as described attcga.cancer.gov/dataportal/data/access/closed/.

Example XIV: Mutation Analysis

Exome Capture and Sequencing was Performed on DNA Isolated from 316HGS-OvCa samples and matched normal samples for each individual. Capturereagents targeted −480,000 exons from −48,500 genes totaling −33megabases of non-redundant sequence. Massively parallel sequencing onthe Illumina GAIIx platform (236 sample pairs) or ABI SOLiD 3 platform(80 sample pairs) yielded −14 gigabases per sample (9×109 bases total).On average, 76% of coding bases were covered in sufficient depth in boththe tumor and matched normal samples to allow confident mutationdetection. 19,356 somatic mutations (−61 per tumor) were annotated andclassified in Table 4. Mutations that may be important in HGS-OvCapathophysiology were identified by (a) searching for non-synonymous orsplice site mutations present at significantly increased frequenciesrelative to background, (b) comparing mutations in this study to thosein COSMIC and OMIM and (c) predicting impact on protein function.

Two different algorithms identified 9 genes (Table 5) for which thenumber of non-synonymous or splice site mutations was significantlyabove that expected based on mutation distribution models. Consistentwith published resultsl3, TP53 was mutated in 303 of 316 samples (283 byautomated methods and 20 after manual review), BRCA 0.1 and BRCA2 hadgermline mutations in 9% and 8% of cases, respectively, and both showedsomatic mutations in an additional 3% of cases. Six other statisticallyrecurrently mutated genes were identified; RB1, NF1. FAT3,CSMD3, GABRA6.and CDKI2. CDKI2 is involved in RNA splicing regulation14 and waspreviously implicated in lung and large intestine tumors. Five of thenine CDK12 mutations were either nonsense or indel, suggesting potentialloss of function, while the four missense mutations (R882L, Y901C,K975E, and L996F) were clustered in its protein kinase domain. GABRA6and FATS both appeared as significantly mutated but did not appear to beexpressed in HGS-OvCa or fallopian tube tissue so it is less likely thatmutation of these genes plays a significant role in HGS-OvCa.

Mutations from this study were compared to mutations in the COSMIC17 andOMIM18 databases to identify additional HGS-OvCa genes that are lesscommonly mutated. This yielded 477 and 211 matches respectivelyincluding mutations in BRAF (N581S), PIK3CA (E545K and H 1047R), KRAS(G12D), and NRAS (Q61R). These mutations have been shown to exhibittransforming activity so we believe that these mutations are rare butimportant drivers in HGS-OvCa.

We combined evolutionary information from sequence alignments of proteinfamilies and whole vertebrate genomes, predicted local protein structureand selected human SwissProt protein features to identify putativedriver mutations using CHASM after training on mutations in knownoncogenes and tumor suppressors. CHASM identified 122 mis-sensemutations predicted to be oncogenic. Mutation-driven changes in proteinfunction were deduced from evolutionary information for all confirmedsomatic missense mutations by comparing protein family sequencealignments and residue placement in known or homology-basedthree-dimensional protein structures using Mutation Assessor.Twenty-seven percent of missense mutations were predicted to impactprotein function.

Example XV: Copy Number Analysis

Somatic copy number alterations (SCNAs) present in the 489 HGS-OvCagenomes were identified and compared with glioblastome multiforme datain FIG. 37A. SCNAs were divided into regional aberrations that affectedextended chromosome regions and smaller focal aberrations. A statisticalanalysis of regional aberrations identified 8 recurrent gains and 22losses, all of which have been reported previously (FIG. 37B). Five ofthe gains and 18 of the losses occurred in more than 50% of tumors.

GISTIC was used to identify recurrent focal SCNAs. This yielded 63regions of focal amplification (FIG. 37C) including 26 that encoded 8 orfewer genes. The most common focal amplifications encoded CCNE1, MYC,and MECOM (FIG. 37C) each highly amplified in greater than 20% oftumors. New tightly-localized amplification peaks in HGS-OvCa encodedthe receptor for activated C-kinase, ZMYND8; the p53 target gene,IRF2BP2; the DNA-binding protein inhibitor, ID4; the embryonicdevelopment gene, PAX8; and the telomerase catalytic subunit, TERT.Three data sources: www.ingenuity.com/, clinicaltrials.gov andwww.drugbank.ca were used to identify possible therapeutic inhibitors ofamplified, over-expressed genes. This search identified 22 genes thatare therapeutic targets including MECOM, MAPK1, CCNE1 and KRAS amplifiedin at least 10% of the cases.

GISTIC also identified 50 focal deletions. The known tumor suppressorgenes PTEN RB1, and NF1 were in regions of homozygous deletions in atleast 2% of tumors. Importantly, RB1 and NF1 also were among thesignificantly mutated genes. One deletion contained only three genes,including the essential cell cycle control gene, CREBBP, which has 5non-synonymous and 2 frameshift mutations.

Example XVI: mRNA and miRNA Expression and DNA Methylation Analysis

Expression measurements for 11,864 genes from three different platforms(Agilent, Affymetrix HuEx, Affymetrix U133A) were combined for subtypeidentification and outcome prediction. Individual platform measurementssuffered from limited, but statistically significant batch effects,whereas the combined data set did not. Analysis of the combined datasetidentified—1,500 intrinsically variable genes that were used for NMFconsensus clustering. This analysis yielded four clusters (FIG. 38A).The same analysis approach applied to a publicly available dataset fromTothill et al., also yielded four clusters. Comparison of the Tothilland TCGA clusters showed a clear correlation. We therefore conclude thatat least four robust expression subtypes exist in HGS-OvCa.

We termed the four HGS-OvCa subtypes Immunoreactive, Differentiated,Proliferative and Mesenchymal based on gene content in the clusters andon previous observations25. T-cell chemokine ligands, CXCL 1I andCXCLIO, and the receptor, CXCR3, characterized the Immunoreactivesubtype. High expression of transcription factors such as HMGA2 andSOX/1, low expression of ovarian tumor markers (MUC1, WIC I 6) and highexpression of proliferation markers such as MCM2 and PCNA defined theProliferative subtype. The Differentiated subtype was associated withhigh expression of MUC I 6 and MUC1 and with expression of the secretoryfallopian tube maker SLP1, suggesting a more mature stage ofdevelopment. High expression of HOX genes and markers suggestive ofincreased stromal components such as for myofibroblasts (FAP) andmicrovascular pericytes (ANGPTL2, ANGPTL1) characterized the Mesenchymalsubtype.

Elevated DNA methylation and reduced tumor expression implicated 168genes as epigenetically silenced in HGS-OvCa compared to fallopian tubecontrols26. DNA methylation was correlated with reduced gene expressionacross all samples. AMT, CCL2I and SPARCLI were noteworthy because theyshowed promoter hypermethylation in the vast majority of the tumors.Curiously, RAB25, previously reported to be amplified and over-expressedin ovarian cancer, also appeared to be epigenetically silenced in asubset of tumors. The BRCA1 promoter was hypermethylated and silenced in56 of 489 (11.5%) tumors as previously reported. Consensus clustering ofvariable DNA methylation across tumors identified four subtypes thatwere significantly associated with differences in age, BRCA inactivationevents, and survival. However, the clusters demonstrated only modeststability.

Survival duration did not differ significantly for transcriptionalsubtypes in the TCGA dataset. The Proliferative group showed a decreasein the rate of MYC amplification and RB1 deletion, whereas theImmunoreactive subtype showed an increased frequency of 3q26.2 (MECOM)amplification. A moderate, but significant overlap between the DNAmethylation clusters and gene expression subtypes was noted(p<2.2*10-16, Chi-square test, Adjusted Rand Index=0.07).

A 193 gene transcriptional signature predictive of overall survival wasdefined using the integrated expression data set from 215 samples. Afterunivariate Cox regression analysis, 108 genes were correlated with poorsurvival, and 85 were correlated with good survival (p-value cutoff of0.01). The predictive power was validated on an independent set of 255TCGA samples as well as three independent expression data sets25,29,30.Each of the validation samples was assigned a prognostic gene score,reflecting the similarity between its expression profile and theprognostic gene signature31 (FIG. 38C). Kaplan-Meier survival analysisof this signature showed statistically significant association withsurvival in all validation data sets (FIG. 38D).

NMF consensus clustering of miRNA expression data identified threesubtypes. Interestingly, miRNA subtype 1 overlapped the mRNAProliferative subtype and miRNA subtype 2 overlapped the mRNAMesenchymalsubtype (FIG. 38D). Survival duration differed significantly betweeniRNA subtypes with patients in miRNA subtype 1 tumors survivingsignificantly longer (FIG. 38e ).

Example XVII: Pathways Influencing Disease

Several analyses integrated data from the 316 fully analyzed cases toidentify biology that contributes to HGS-OvCa. Analysis of the frequencywith which known cancer-associated pathways harbored one or moremutations, copy number changes, or changes in gene expression showedthat the RB1 and PI3K/RAS pathways were deregulated in 67% and 45% ofcases, respectively (FIG. 39A). A search for altered subnetworks in alarge protein-protein interaction network32 using HotNet33 identifiedseveral known pathways, including the Notch signaling pathway, which wasaltered in 23% of HGS-OvCa samples (FIG. 39B).

Published studies have shown that cells with mutated or methylated BRCA1or mutated BRCA2 have defective homologous recombination (FIR) and arehighly responsive to PARP inhibitors 35-37. FIG. 39C shows that 20% ofHGS-OvCa have germline or somatic mutations in BRCA1/2, that 11% havelost BRCA1 expression through DNA hypennethylation and that epigeneticsilencing of BRCA1 is mutually exclusive of BRCA1/2 mutations(P=4.4×10-4, Fisher's exact test). Univariate survival analysis of BRCAstatus (FIG. 39C) showed better overall survival (OS) for BRCA mutatedcases than BRCA wild-type cases. Interestingly, epigenetically silencedBRCA1 cases exhibited survival similar to BRCA1/2 WT HGS-OvCa (median OS41.5 v. 41.9 months, P=0.69, log-rank test). This suggests that BRCA1 isinactivated by mutually exclusive genomic and epigenomic mechanisms andthat patient survival depends on the mechanism of inactivation. Genomicalterations in other HR genes that might render cells sensitive to PARPinhibitors discovered in this study include amplification or mutation ofEMSY (8%), focal deletion or mutation of PTEN (7%); hypermethylation ofRAD51C (3%), mutation of ATM-ATR (2%), and mutation of Fanconi Anemiagenes (5%). Overall, HR defects may be present in approximately half ofHGS-OvCa, providing a rationale for clinical trials of PARP inhibitorstargeting tumors these HR-related aberrations.

Comparison of the complete set of BRCA inactivation events to allrecurrently altered copy number peaks revealed an unexpectedly lowfrequency of CCNE1 amplification in cases with BRCA inactivation (8% ofBRCA altered cases had CCNE1 amplification v. 26% of BRCA wild typecases, FDR adjusted P=0.0048). As previously reported39, overallsurvival tended to be shorter for patients with CCNE1 amplificationcompared to all other cases (P=0.072, log-rank test). However, nosurvival disadvantage for CCNE1-amplified cases (P=0.24, log-rank test)was apparent when looking only at BRCA wild-type cases, suggesting thatthe previously reported CCNE1 survival difference can be explained bythe better survival of BRCA-mutated cases.

Finally, a probabilistic graphical model (PARADIGM40) searched foraltered pathways in the NCI Pathway Interaction Database identifying theFOXM1 transcription factor network (FIG. 39D) as significantly alteredin 87% of cases. FOXM1 and its proliferation-related target genes; AURB,CCNE1, BIRC5, CDC25, and PLK1, were consistently over-expressed but notaltered by DNA copy number changes, indicative of transcriptionalregulation. TP53 represses FOXM1 following DNA damage42, suggesting thatthe high rate of TP53 mutation in HGS-OvCa contributes to FOXM1overexpression. In other datasets, the FOXM1 pathway is significantlyactivated in tumors relative to adjacent epithelial tissue and isassociated with HGS-OvCa.

Example XVIII: Frequently Altered Pathways in Ovarian Serous Carcinomas

To identify significantly altered pathways through an integratedanalysis of both copy number and gene expression, we applied PARADIGM.The computational model incorporates copy number changes, geneexpression data, and pathway structures to produce an integrated pathwayactivity (IPA) for every gene, complex, and genetic process present inthe pathway database. We use the term “entity” to refer to any moleculein a pathway be it a gene, complex, or small molecule. The IPA of anentity refers only to the final activity. For a gene, the IPA onlyrefers to the inferred activity of the active state of the protein,which is inferred from copy number, gene expression, and the signalingof other genes in the pathway. We applied PARADIGM to the ovariansamples and found alterations in many different genes and processespresent in pathways contained in the National Cancer Institutes' PathwayInteraction Database (NCI-PID). We assessed the significance of theinferred alterations using 1000 random simulations in which pathwayswith the same structure were used but arbitrary genes were assigned atdifferent points in the pathway. In other words, one random simulationfor a given pathway kept the set of interactions fixed so that anarbitrary set of genes were connected together with the pathway'sinteractions. The significance of all samples' IPAs was assessed againstthe same null distribution to obtain a significance level for eachentity in each sample. IPAs and the percentage of samples in which theyare significant and IPAs with a standard deviation of at least 0.1 aredisplayed as a heatmap in FIG. 28.

Table 3 shows the pathways altered by at least three standard deviationswith respect to permuted samples found by PARADIGM. The FOXM1transcription factor network was altered in the largest number ofsamples among all pathways tested—67% of entities with alteredactivities when averaged across samples. In comparison, pathways withthe next highest level of altered activities in the ovarian cohortincluded PLK1 signaling events (27%), Aurora B signaling (24%), andThromboxane A2 receptor signaling (20%). Thus, among the pathways inNCI-PID, the FOXM1 network harbors significantly more altered activitiesthan other pathways with respect to the ovarian samples.

The FOXM1 transcription factor network was found to be differentiallyaltered in the tumor samples compared to the normal controls in thehighest proportion of the patient samples (FIG. 29). FOXM1 is amultifunctional transcription factor with three known dominant spliceforms, each regulating distinct subsets of genes with a variety of rolesin cell proliferation and DNA repair. The FOXMIc isoform directlyregulates several targets with known roles in cell proliferationincluding AUKB, PLK1, CDC25, and BIRC5. On the other hand, the FOXM1bisoform regulates a completely different subset of genes that includethe DNA repair genes BRCA2 and XRCC I. CHEK2, which is under indirectcontrol of ATM, directly regulates FOXM 1 s expression level.

We asked whether the IPAs of the FOXM1 transcription factor itself weremore highly altered than the IPAs of other transcription factors. Wecompared the FOXM1 level of activity to all of the other 203transcription factors in the NCI-PID. Even compared to othertranscription factors in the NCI set, the FOXM1 transcription factor hadsignificantly higher levels of activity (p<0.0001; K-S test) suggestingfurther that it may be an important signature (FIGS. 30A-30B).

Because FOXM1 is also expressed in many different normal tissues ofepithelial origin, we asked whether the signature identified by PARADIGMwas due to an epithelial signature that would be considered normal inother tissues. To answer this, we downloaded an independent dataset fromGEO (GSE10971) in which fallopian tube epithelium and ovarian tumortissue were microdissected and gene expression was assayed. We foundthat the levels of FOXM1 were significantly higher in the tumor samplescompared to the normals, suggesting FOXM1 regulation is indeed elevatedin cancerous tissue beyond what is seen in normal epithelial tissue(FIG. 31).

Because the entire cohort for the TCGA ovarian contained samples derivedfrom high-grade serous tumors, we asked whether the FOXM1 signature wasspecific to high-grade serous. We obtained the log expression of FOXM1and several of its targets from the dataset of Etemadmoghadam et al.(2009) in which both low- and high-grade serous tumors had beentranscriptionally profiled. This independent data confirmed that FOXM 1and several of its targets are significantly up-regulated in serousovarian relative to low-grade ovarian cancers (FIG. 32). To determine ifthe 25 genes in the FOXM1 transcription factor network contained asignificant proportion of genes with higher expression in high-gradedisease, we performed a Student's t-test using the data fromEtemadmoghadam. 723 genes in the genome (5.4%) were found to besignificantly up-regulated in high- versus low-grade cancer at the 0.05significance level (corrected for multiple testing using theBenjamini-Hochberg method). The FOXM1 network was found to have 13 ofits genes (52%) differentially regulated, which is a significantproportion based on the hypergeometric test (P<3.8*10⁻¹²). Thus, highexpression of the FOXM1 network genes does appear to be specificallyassociated with high-grade disease when compared to the expression oftypical genes in the genome.

FOXM1's role in many different cancers including breast and lung hasbeen well documented but its role in ovarian cancer has not beeninvestigated. FOXM1 is a multifunctional transcription factor with threeknown splice variants, each regulating distinct subsets of genes with avariety of roles in cell proliferation and DNA repair. An excerpt ofFOXM1's interaction network relevant to this analysis is shown as FIG.27. The FOXMla isoform directly regulates several targets with knownroles in cell proliferation including AUKB, PLK1, CDC25, and BIRC5. Incontrast, the FOXM1b isoform regulates a completely different subset ofgenes that include the DNA repair genes BRCA2 and XRCC1. CHEK2, which isunder indirect control of ATM, directly regulates FOXM1's expressionlevel. In addition to increased expression of FOXM1 in most of theovarian patients, a small subset also have increased copy numberamplifications detected by CBS (19% with copy number increases in thetop 5% quantile of all genes in the genome measured). Thus thealternative splicing regulation of FOXM1 may be involved in the controlswitch between DNA repair and cell proliferation. However, there isinsufficient data at this point to support this claim since the exonstructure distinguishing the isoforms and positions of the Exon arrayprobes make it difficult to distinguish individual isoform activities.Future high-throughput sequencing of the mRNA of these samples may helpdetermine the differential levels of the FOXM1 isoforms. The observationthat PARADIGM detected the highest level of altered activity centered onthis transcription factor suggests that FOXM1 resides at a criticalregulatory point in the cell.

Example XIX: Data Sets and Pathway Interactions

Both copy number and expression data were incorporated into PARADIGMinference. Since a set of eight normal tissue controls was available foranalysis in the expression data, each patient's gene-value wasnormalized by subtracting the gene's median level observed in the normalfallopian control. Copy number data was normalized to reflect thedifference in copy number between a gene's level detected in tumorversus a blood normal. For input to PARADIGM, expression data was takenfrom the same integrated dataset used for subtype analysis and the copynumber was taken from the segmented calls of MSKCC Agilent 1M copynumber data.

A collection of pathways was obtained from NCI-PID containing 131pathways, 11,563 interactions, and 7,204 entities. An entity ismolecule, complex, small molecule, or abstract concept represented as“nodes” in PARADIGM's graphical model. The abstract concepts correspondto general cellular processes (such as “apoptosis” or “absorption oflight,”) and families of genes that share functional activity such asthe RAS family of signal transducers. We collected interactionsincluding protein-protein interactions, transcriptional regulatoryinteractions, protein modifications such as phosphorylation andubiquitinylation interactions.

Example XX: Inference of Integrated Molecular Activities in PathwayContext

We used PARADIGM, which assigns an integrated pathway activity (IPA)reflecting the copy number, gene expression, and pathway context of eachentity.

The significance of IPAs was assessed using permutations of gene- andpatient-specific cross-sections of data. Data for 1000 “null” patientswas created by randomly selecting a gene-expression and copy number pairof values for each gene in the genome. To assess the significance of thePARADIGM IPAs, we constructed a null distribution by assigning randomgenes to pathways while preserving the pathway structure.

Example XXI: Identification of FOXM1 Pathway

While all of the genes in the FOXM1 network were used to assess thestatistical significance during the random simulations, in order toallow visualization of the FOXM1 pathway, entities directly connected toFOXM1 with significantly altered IPAs according to FIG. 29 were chosenfor inclusion in FIG. 27. Among these, genes with roles in DNA repairand cell cycle control found to have literature support for interactionswith FOXM1 were displayed. BRCC complex members, not found in theoriginal NCI-PID pathway, were included in the plot along with BRCA2,which is a target of FOXM1 according to NCI-PID. Upstream DNA repairtargets were identified by finding upstream regulators of CBEK2 in otherNCI pathways (for example, an indirect link from ATM was found in thePLK3 signaling pathway).

Example XXII: Clustering

The use of inferred activities, which represent a change in probabilityof activity and not activity directly, it enables entities of varioustypes to be clustered together into one heatmap. To globally visualizethe results of PARADIGM inference, Eisen Cluster 3.0 was used to performfeature filtering and clustering. A standard deviation filtering of 0.1resulted in 1598 out of 7204 pathway entities remaining, and averagelinkage, uncentered correlation hierarchical cluster was performed onboth the entities and samples.

Example XXIII: Cell Lines Model Many Important Tumor Subtypes andFeatures

The utility of cell lines for identification of clinically relevantmolecular predictors of response depends on the extent to which thediverse molecular mechanisms that determine response in tumors areoperative in the cell lines. We reported previously on similaritiesbetween cell line models and primary tumors at both transcript andgenome copy number levels' and we refine that comparison here usinghigher resolution platforms and analysis techniques. Specifically, weused hierarchical consensus clustering (HCC) of gene expression profilesto classify 50 breast cancer cell lines and 5 non-malignant breast celllines into three transcriptional subtypes: luminal, basal and the newlydescribed claudin-low (FIG. 14A). These subtypes are refined versions ofthose described earlier, where basal and caludin-low maps to thepreviously designated basal A and basal B subtypes, respectively, Table7. A refined high-resolution SNP copy number analysis (FIG. 14B)confirms that the cell line panel models regions of recurrentamplification at 8q24 (MYC), 11q13 (CCND1), 17q12 (ERBB2), 20q13(STK15/AURKA), and homozygous deletion at 9p21 (CDKN2A) found in primarytumors. Given the clinical relevance of the ERBB2 tumor subtype asdetermined by trastuzumab and lapatinib therapy, we examined cell lineswith DNA amplification of ERBB2 as a special subtype designatedERBB2^(AMP). Overall, our identification of luminal, basal, claudin-lowand ERBB2″^(P) cell lines is consistent with the clinical biology.

Example XIX: The Cell Lines Exhibit Differential Sensitivities to MostTherapeutic Compounds

We examined the sensitivity of our cell line panel to 77 therapeuticcompounds. We used a cell growth assay with a quantitative endpointmeasured after three days of continuous exposure to each agent at nineconcentrations. The anti-cancer compounds tested included a mix ofconventional cytotoxic agents (for example, taxanes, platinols,anthracylines) and targeted agents (for example, SERMs and kinaseinhibitors). In many cases, several agents targeted the same protein ormolecular mechanism of action. We determined a quantitative measure ofresponse for each compound as the concentration required to inhibitgrowth by 50% (designated the GI₅₀), In cases where the underlyinggrowth data are of high quality, but 50% inhibition was not achieved, weset GI₅₀ to the highest concentration tested. GI₅₀ values are providedin Table 8 for all compounds. We excluded three compounds (PS1145,cetuximab and baicalein) from further analysis because the variabilityin cell line response was minimal.

A representative waterfall plot illustrating the variation in responseto the Sigma AKT1-2 inhibitor along with associated transcriptionalsubtypes is shown in FIG. 10A. Sensitivity to this compound is highestin luminal and ERBB2^(AmP) and lower in basal and claudin-low breastcancer cell lines. Waterfall plots showing the distribution of GI₅₀values among the cell lines for all compounds are in the SupplementaryAppendix. We established the reproducibility of the overall data set bycomputing the median absolute deviation of GI₅₀ values for 229compound/cell line combinations with 3 or 4 replicates. The medianaverage deviation was 0.15 across these replicates (FIGS. 15A-15B). Weassessed concordance of response to 8 compounds by computing thepairwise Pearson's correlation between sets of 0150 values (FIG. 15B.Sensitivities for pairs of drugs with similar mechanisms of action werehighly correlated, suggesting similar modes of action.

Example XX: Many Compounds were Preferentially Effective in Subsets ofthe Cell Lines

A central premise of this study is that associations between responsesand molecular subtypes observed in preclinical cell line analyses willbe recapitulated in the clinic in instances where the predictivemolecular features in the cell lines are mirrored in human tumors. Weestablished response-subtype associations by using non-parametric ANOVAsto compare GI₅₀ values across transcriptional and genomics subtypes.

Overall, 33 of 74 compounds tested showed transcription subtype-specificresponses (FDR p<0.2, Table 7 and Table 9). FIG. 10C shows ahierarchical clustering of the 34 agents with significant associationswith one or more of the luminal, basal, claudin-low and ERBB2^(AmP)subtypes. The 11 agents most strongly associated with subtype wereinhibitors of receptor tyrosine kinase signaling and histone deacetylaseand had the highest efficacy in luminal and/or ERBB2′ cell lines. Thethree next most subtype-specific agents—etoposide, cisplatin, anddocetaxel—show preferential activity in basal and/or claudin-low celllines as observed clinically. Agents targeting the mitotic apparatus,including ixabepilone, GSK461364 (polo kinase inhibitor) and GSK1070916(aurora kinase inhibitor) also were more active against basal andclaudin-low cell lines. AG1478, BIBW2992 and gefitinib, all of whichtarget EGFR and/or ERBB2 were positively associated with ERBB2amplification. Geldanamycin, an inhibitor of HSP90 also was positivelyassociated with ERBB2 amplification. Interestingly, VX-680 (aurorakinase inhibitor) and CGC-11I44 (polyamine analogue) both werenegatively associated with ERBB2 amplification indicating that these arerelatively poor therapies for ERBB2^(ANW) tumors.

We identified 7 associations (6 unique compounds) between response andrecurrent focal high-level copy number aberrations (CNAs; samplet-tests, FDR p<0.2, Table 10). FIG. 10D shows that (a) Homozygousdeletion at 9p21 (CDKN2A and CDKN2B) was associated with response tovinorelbine, ixabepilone and fascalypsin. Fascalypsin inhibited CDK4 andthis specificity is consistent with the role of the, _(6INK4A) productof CDKN2A in inhibiting CDK4²⁰. (b) Amplification at 20q13 (whichencodes AURKA), was associated with resistance, rather than sensitivity,to GSK1070916 and VX-680 which target A URKB and AURKC²³. This suggeststhat amplification of AURKA provides a bypass mechanism for A URKB andAURKC inhibitors. (c) Amplification at 11q13 (CCND1) was associated withsensitivity to carboplatin and the AURKB/C inhibitor GSK1070916.

Example XXI: Subtype Specificity Dominates Growth Rate Effects

In general, we found that luminal subtype cell lines grew more slowlythan basal or claudin-low cells (Kruskal-Wallis test p=0.006, FIG. 16Aand Table 7) and the range of doubling times was broad (18 to 300hours). This raised the possibility that the most sensitive cell lineswere those that grew most rapidly. If so, then the observed associationsto subtype could represent an association to a covariate. We tested thishypothesis by assessing the effects of subtype and doubling timesimultaneously using Analysis of Covariance (ANCOVA) and found that 22of the 33 subtype-specific compounds had better associations withsubtype than with doubling time (mean log ratio of p-values=0.92,standard deviation 1.11). This supports the idea that subtype membershipis a better predictor of response than growth rate. Moreover, 15 of 33subtype-specific compounds were more effective in the more slowlygrowing luminal cell lines (Table 7). One agent, 5-florouracil, was notsignificant in the subtype test alone but showed strong significance inthe ANCOVA model for both class and doubling time. The response to5-florouracil decreased as doubling time increased in both luminal andbasal cell lines (FIG. 16B). We conclude that in most cases, the 3-daygrowth inhibition assay is detecting molecular signature-specificresponses that are not strongly influenced by growth rate.

Example XXII: Integration of Copy Number and Transcription MeasurementsIdentifies Pathways of Subtype Specific Responses

We used the network analysis tool PARADIGM²⁴ to identify differences inpathway activity among the subtypes in the cell line panel. The analysisis complicated by the fact that the curated pathways are partiallyoverlapping. For example EGFR, PI3 kinase and MEK are often curated asseparate pathways when in fact they are components of a single largerpathway. To address this issue, PARADIGM merges approximately 1400curated signal transduction, transcriptional and metabolic pathways intoa single superimposed pathway (SuperPathway) to eliminate suchredundancies. Using both the copy number and gene expression data for aparticular cell line, PARADIGM uses the pathway interactions to inferintegrated pathway levels (IPLs) for every gene, complex, and cellularprocess.

We compared cell lines to primary breast tumors by their pathwayactivations using the PARADIGM IPLs. Data for the cell line-tumorcomparison was carried out using data generated by The Cancer GenomeAtlas (TCGA) project (cancergenome.nih.gov). FIG. 11 shows pathwayactivities for each tumor and cell line after hierarchical clustering.The top five pathway features for each subtype are listed in Table 11.Overall, the tumors and cell line subtypes showed similar pathwayactivities and the deregulated pathways were better associated withtranscriptional subtype than origin (FIGS. 13A-13B). However, pathwaysassociated with the claudin low cell line subtype are not wellrepresented in the tumor—possibly because the claudin-low subtype isover-represented in the cell line collection and the luminal. A subtypeis missing (FIGS. 12A-12D).

Example XXIII: Identification of Subtype-Specific Pathway Markers

We asked whether intrinsic pathway activities underlie the differencesbetween the subtypes. To this end, we identified subnetworks of theSuperPathway containing gene activities differentially up- ordown-regulated in cell lines of one subtype compared to the rest.Comparison of pathway activities between basal cell lines and all othersin the collection identified a network comprised of 965 nodes connectedby 941 edges, where nodes represent proteins, protein complexes, orcellular processes and edges represent interactions, such as proteinphosphorylation, between these elements (see FIGS. 18-22). FIG. 35Ashows upregulation of the MYC/MAX subnetwork associated withproliferation, angiogenesis, and oncogenesis; and upregulation of theERK1/2 subnetwork controlling cell cycle, adhesion, invasion, andmacrophage activation. The FOXM1 and DNA damage subnetworks also weremarkedly upregulated in the basal cell lines. Comparison of theclaudin-low subtype with all others showed upregulation of many of thesame subnetworks as in basal cell lines with some exceptions, includingupregulation of the beta-catenin (CTNNB I) network in claudin low celllines as compared to the basal cells (FIG. 35B). Beta-catennin has beenimplicated in tumorigenesis, and is associated with poor prognosis.Comparison of the luminal cell lines with all others showeddown-regulation of an ATF2 network, which inhibits tumorigenicity inmelanoma, and up-regulation of FOXA1/FOXA2 networks that controltranscription of ER-regulated genes and are implicated in good prognosisluminal breast cancers (FIG. 35C). Comparison of ERBB2^(AmP) cell lineswith all others showed many network features common to luminal cells—notsurprising because most ERBB2^(AmP) cells also are classified as luminalcells. However, FIG. 35D shows down regulation centered on RPS6KBP1 inERBB2^(Asc) cell lines.

Comparative analysis of differential drug response among the cell linesusing the IPLs revealed pathway activities that provide informationabout mechanisms of response. For example, the basal cell lines arepreferentially sensitive to cisplatin, a DNA damaging agent, and alsoshowed upregulation of a DNA-damage response subnetwork that includesATM, CHEK1 and BRCA1, key players associated with response to cisplatin(FIG. 36A). Likewise, ERBB2^(AmP) cell lines are sensitive togeldanamycin, an inhibitor of HSP90, and also showed up-regulation inthe ERBB2-HSP90 subnetwork (FIG. 36B). This observation is consistentwith the mechanism of action for geldanamycin: it binds ERBB2 leading toits degredation. We found that the ERBB2^(AmP) cell lines were resistantto the aurora kinase inhibitor VX-680, and further that sensitivity tothis compound was not associated with amplification at 20q13 (AURKA).This raises the possibility that this resistance may be mediated throughCCNB I, which is co-regulated with AURKB by FOXM1. Of the four subtypes,ERBB2^(AmP) is the only one that shows substantial down-regulation ofCCNB1 (FIGS. 22A-22C). This proposed mechanism is supported by theobservation that in primary tumors, CCNB1 gene expression issignificantly correlated with AURKB gene expression.

Example XXIV: Cell Growth Inhibition Assay and Growth Rate

We assessed the efficacy of 77 compounds in our panel of 55 breastcancer cell lines. This assay was performed as previously described(Kuo, W. L. et al. A systems analysis of the chemosensitivity of breastcancer cells to the polyamine analogue PG-11047. BMC Med 7, 77,doi:1741-7015-7-77 [pii] 10.1186/1741-7015-7-77 (2009)). Briefly, cellswere treated for 72 hours with a set of 9 doses of each compound in 1:5serial dillution. Cell viability was determined using the Cell Titer Gloassay. Doubling time (DT) was estimated from the ratio of 72 h to Oh foruntreated wells.

We used nonlinear least squares to fit the data with a Gompertz curvewith the following parameters: upper and lower asymptotes, slope andinflection point. The fitted curve was transformed into a GI curve usingthe method described by the NC1/NIH DTP Human Tumor Cell Line ScreenProcess and previously described (Screening Services—NCI-60 DTP HumanTumor Cell Line Screen. dtp.ncl.nih.gov/branchsibtbflvisp.html. Monks,A. et al. Feasibility of a high-flux anticancer drug screen using adiverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83,757-766 (1991)).

We assessed a variety of response measures including the compoundconcentration required to inhibit growth by 50% (GI₅₀), theconcentration necessary to completely inhibit growth (Total GrowthInihibition, TGI) and the concentration necessary to reduce thepopulation by 50% (Lethal Concentration 50%, LC₅₀). In cases where theunderlying growth data are of high quality, but the end point response(GI₅₀, TG1, LC₅₀) was not reached, the values were set to the highestconcentration tested. GI₅₀ represents the first threshold reached, andtherefore contains the most accurate set of measurements.

The drug response data was filtered to meet the following criteria: 1)median standard deviation across the 9 triplicate datapoints <0.20; 2)DT+1-2SD of the median DT for a particular cell line; 3) slope of thefitted curve >0.25; 4) growth inhibition at the maximum concentration<50% for datasets with no clear response. Approximately 80% of the drugplates pass all filtering requirements. We used the median absolutedeviation (MAD), a robust version of standard deviation, to assess thereliability of our replicate measures of GI₅₀. Curve fitting andfiltering were performed with custom-written R packages.

Example XXV: Drug Screening

Each drug included in the statistical analysis satisfied the followingscreening criteria for data quality: 1) Missing values: No more than 40%of GI₅₀ values can be missing across the entire set of cell lines; 2)Variability: For at least 3 cell lines, either GI₅₀>1.5. mGI₅₀ orGI₅₀<0.5. mGI₅₀, where mGI₅₀ is the median GIso for a given drug.Compounds failing these criteria were excluded from analysis.

Example XXVI: SNP Array and DNA Copy Number Analysis

Affymetrix Genome-Wide Human SNP Array 6.0 was used to measure DNA copynumber data. The array quality and data processing was performed usingthe R statistical framework (www.r-projectorg.) based aroma.affymetrix.The breast cancer cell line SNP arrays were normalized using 20 normalsample arrays as described (Bcngtsson, H., Irizarry, R., Carvalho, B. &Speed, T. P. Estimation and assessment of raw copy numbers at the singlelocus level. Bioinformatics (Oxford, England) 24, 759-767 (2008)). Datawere segmented using circular binary segmentation (CBS) from thebioconductor package DNAcopy (Olshen, A. B., Venkatraman, E. S., Lucito,R. & Wigler, M. Circular binary segmentation for the analysis ofarray-based DNA copy number data. Biostatistics (Oxford, England) 5,557-572 (2004)). Significant DNA copy number changes were analyzed usingMATLAB based Genomic Identification of Significant Targets in Cancer(GISTIC) (Beroukhim, R. et al. Assessing the significance of chromosomalaberrations in cancer: methodology and application to glioma. Proc NatlAcrd Sci USA 104, 20007-20012 (2007)). Raw data are available in TheEuropean Genotype Archive (EGA) with accession number, EGAS00000000059.

In order to ensure the greatest chance at detecting significant changesin copy number, we omitted the non-malignant cell lines from the GISTICanalysis. GISTIC scores for one member of each isogenic cell line pairwas used to infer genomic changes in the other: AU565 was inferred fromSKBR3; HCC1500 was inferred from HCC1806; LY2 was inferred from MCF7;ZR75B was inferred from ZR751.

Example XXVII: Exon Array Analysis

Gene expression data for the cell lines were derived from AffymetrixGeneChip Human Gene 1.0 ST exon arrays. Gene-level summaries ofexpression were computed using the aroma.affymetrix R package, withquantile normalization and a log-additive probe-level model (PLM) basedon the “HuEx-_0-st-v2,core” chip type. Transcript identifiers wereconverted to HGNC gene symbols by querying the Ensembl database usingthe BioMart R package. The resulting expression profiles weresubsequently filtered to capture only those genes expressing a standarddeviation greater than 1.0 on the log_(e)-scale across all cell lines.The raw data are available in ArrayExpress (E-MTAB-181).

Example XXVIII: Consensus Clustering

Cell line subtypes were identified using hierarchical consensusclustering (Monti, S., Tamayo, P., Mesirov, J. P. & Golub, T. A.Consensus Clustering: A Resampling-Based Method for Class Discovery andVisualization of Gene Expression Microarray Data. Machine Learning 52,91-118 (2003). Consensus was computed using 500 samplings of the celllines, 80% of the cell lines per sample, agglomerative hierarchicalclustering, Euclidean distance metric and average linkage.

Example XXIX: Associations of Clinically Relevant Subtypes and Responseto Therapeutic Agents

We used three schemes to compare GI₅₀s: 1) luminal vs. basal vs.claudin-low; 2) luminal vs. basal+claudin-low; and 3) ERBB2-AMP vs.non-ERBB2-AMP. Differences between G150s of the groups were comparedwith a non-parametric ANOVA or t-test, as appropriate, on the ranks. Wecombined the p-values for the three sets of tests and used falsediscovery rate (FDR) to correct for multiple testing. For thethree-sample test, we performed a post-hoc analysis on the compoundswith a significant class effect by comparing each group to all others todetermine which group was most sensitive. The p-values for the post-hoctest were FDR-corrected together. In all cases, FDR p<0.20 was deemedsignificant. If it was the case that the basal+claudin-low group wasfound to be significant in scheme 2, but only one of these groups wassignificant in scheme 1, we gave precedence to the 3 sample case whenassigning class specificity. Analyses were performed in R.

Example XXX: Association of Genomic Changes and Response to TherapeuticAgents

We used a t-test to assess the association between recurrent copy numberchanges (at 8q24 (MYC), 11q13 (CCND1), 20q13 (STK15/AURKA)) and drugsensitivity. We combined into a single group cell lines with low or noamplification and compared them to cell lines with high amplification.The comparable analysis was performed for regions of deletion. Celllines for which the GI₅₀ was equal to the maximum concentration testedwere omitted from analysis. We omitted compounds where any group hadfewer than five samples.

Example XXXI: Association of Growth Rate and Response to TherapeuticAgents

To assess the effects of cell line class and growth rate on drugsensitivity, we performed a set of 2-way Analysis of Covariance (ANCOVA)tests, one for each of the three cell line classification schemesdescribed above. This yielded six sets of p-values (2 main effects×3classification schemes); we used a single FDR correction to assesssignificance, and declared FDR p-values<0.20 to be of interest. Weperformed these analyses in R with the functions Im and ANOVA, which isavailable as part of the car package.

Example XXXII: Integrated Pathway Analysis

Integration of copy number, gene expression, and pathway interactiondata was performed using the PARADIGM software. Briefly, this procedureinfers integrated pathway levels (IPLs) for genes, complexes, andprocesses using pathway interactions and genomic and functional genomicdata from a single cell line or patient sample. See Example XL fordetails.

Example XXXIII: TCGA and Cell Line Clustering

We asked whether the activities inferred for the cell lines clusteredwith their respective subtypes in the TCGA tumor samples. To avoidbiases caused by highly connected hub genes and highly correlatedactivities, cell lines and tumor samples were clustered using a set of2351 non-redundant activities determined by a correlation analysis (seeSupplemental Methods). The degree to which cell lines clustered withtumor samples of the same subtype was calculated using aKolmogorov-Smimov test to compare a distribution of t-statisticscalculated from correlations between pairs of cell lines and tumorsamples of the same subtype to a distribution calculated from cell linepairs of different subtypes (see Supplemental Methods). See Example XLIfor details.

Example XXXIV: Identification of Subtype Pathway Markers

We searched for interconnected genes that collectively show differentialactivity with respect to a particular subtype. Each subtype was treatedas a dichotomization of the cell lines into two groups: one groupcontained the cell lines belong to the subtype and the second groupcontained the remaining cell lines. We used the R implementation of thetwo-class Significance Analysis of Microarrays (SAM) algorithm (Tusher,V. G., Tibshirani, R. & Chu, G. Significance analysis of microarraysapplied to the ionizing radiation response. Proc Natl Acad Sci USA 98,5116-5121, doi:10.1073/pnas.091062498 [pii] (2001)) to compute adifferential activity (DA) score for each concept in the SuperPathway.For subtypes, positive DA corresponds to higher activity in the subtypecompared to the other cell lines.

The coordinated up- and down-regulation of closely connected genes inthe SuperPathway reinforces the activities inferred by PARADIGM. If theactivities of neighboring genes are also correlated to a particularphenotype, we expect to find entire subnetworks with high DA scores. Weidentified regions in the SuperPathway in which concepts of highabsolute DA were interconnected by retaining only those links thatconnected two concepts in which both concepts had DA scores higher thanthe average absolute DA.

Example XXXV: Integrated Pathway Analysis

Integration of copy number, gene expression, and pathway interactiondata was performed using the PARADIGM software²⁴. Briefly, thisprocedure infers integrated pathway levels (IPLs) for genes, complexes,and processes using pathway interactions and genomic and functionalgenomic data from a single cell line or patient sample. TCGA BRCA datawas obtained from the TCGA DCC on Nov. 7, 2010. TCGA and cell line geneexpression data were median probe centered within each data setseparately. All of the values in an entire dataset (either the celllines or TCGA tumor samples), were rank transformed and converted to−log 10 rank ratios before supplying to PARADIGM. Pathways were obtainedin BioPax Level 2 format from pid.nci.nih.gov/ and included NCI-PID,Reactome, and BioCarta databases. Interactions were combined into amerged Superimposed Pathway (SuperPathway). Genes, complexes, andabstract processes (for example, “cell cycle”) were retained as pathwayconcepts. Before merging gene concepts, all gene identifiers weretranslated into HUGO nomenclature. All interactions were included and noattempt was made to resolve conflicting influences. A breadth-firstundirected traversal starting from P53 (the most connected component)was performed to build one single component. The resulting mergedpathway structure contained a total of 8768 concepts representing 3491proteins, 4757 complexes, and 520 processes. Expectation-Maximizationparameters for PARADIGM were trained on the cell line data and thenapplied to the TCGA samples. Data from the cell lines and tumor sampleswere then combined into a single data matrix. Any entry without at least1 value above 0.5 IPL in either the data from cell lines or tumorsamples was removed from further analysis.

Example XXXVI: TCGA and Cell Line Clustering

Using PARADIGM IPLs, cell lines were clustered together with TCGA tumorsamples to determine if cell lines were similar to tumor samples of thesame subtype. Well-studied areas of the SuperPathway contain genes withmany interactions (hubs) and large signaling chains of many intermediatecomplexes and abstract processes for which no direct data is available.To avoid bias toward hubs, pathway concepts with highly correlatedvectors (Pearson correlation coefficient >0.9) across both the cell lineand tumor samples were unified into a single vector prior to clustering.This unification resulted in 2351 non-redundant vectors from theoriginal 8939 pathway concepts.

Samples were clustered using the resulting set of non-redundantconcepts. The matrix of inferred pathway activities for both the 47 celllines and 183 TCGA tumor samples was clustered using complete linkagehierarchical agglomerative clustering implemented in the Eisen Clustersoftware package version 3.0 Uncentered Pearson correlation was used asthe metric for the pathway concepts and Euclidean distance was used forsample metric.

To quantify the degree to which cell lines clustered with tumor samplesof the same subtype, we compared two distributions oft-statisticsderived from Pearson correlations. Let C₅ be the set of cell lines ofsubtypes. Similarly, let T, be the set of TCGA tumor samples of subtypes. For example, C_(basai) and T_(basai) are the set of all basal celllines and basal tumor samples respectively. The first distribution wasmade up of t-statistics derived from the Pearson correlations betweenevery possible pair containing a cell line and tumor sample of the samesubtype; i.e. for all subtypes s, every pairwise correlationt-statistics was computed between a pair (a, h) such that a F C, and b FT. The second distribution was made of correlation t-statistics betweencell lines of different subtypes; that is, computed over pairs (a, b)such that a E C., and b E c* and s s′. We performed a Kolmogorov-Smirnovtest to compare the distributions.

Example XXXVII: Integrated Pathway Analysis

Integration of copy number, gene expression, and pathway interactiondata was performed using the PARADIGM software. Briefly, this procedureinfers integrated pathway levels (IPLs) for genes, complexes, andprocesses using pathway interactions and genomic and functional genomicdata from a single cell line or patient sample. TCGA BRCA data wasobtained from the TCGA DCC on Nov. 7, 2010. TCGA and cell line geneexpression data were median probe centered within each data setseparately. All of the values in an entire dataset (either the celllines or TCGA tumor samples), were rank transformed and converted to−log 10 rank ratios before supplying to PARADIGM. Pathways were obtainedin BioPax Level 2 format on Oct. 13, 2010 from pid.nci.nih.gov/ andincluded NCI-PID, Reactome, and BioCarta databases. Interactions werecombined into a merged Superimposed Pathway (SuperPathway). Genes,complexes, and abstract processes (for example, “cell cycle”) wereretained as pathway concepts. Before merging gene concepts, all geneidentifiers were translated into HUGO nomenclature. All interactionswere included and no attempt was made to resolve conflicting influences.A breadth-first undirected traversal starting from P53 (the mostconnected component) was performed to build one single component. Theresulting merged pathway structure contained a total of 8768 conceptsrepresenting 3491 proteins, 4757 complexes, and 520 processes.Expectation-Maximization parameters for PARADIGM were trained on thecell line data and then applied to the TCGA samples. Data from the celllines and tumor samples were then combined into a single data matrix.Any entry without at least 1 value above 0.5 IPL in either the data fromcell lines or tumor samples was removed from further analysis.

Example XXXVIII: TCGA and Cell Line Clustering

Using PARADIGM IPLs, cell lines were clustered together with TCGA tumorsamples to determine if cell lines were similar to tumor samples of thesame subtype. Well-studied areas of the SuperPathway contain genes withmany interactions (hubs) and large signaling chains of many intermediatecomplexes and abstract processes for which no direct data is available.To avoid bias toward hubs, pathway concepts with highly correlatedvectors (Pearson correlation coefficient >0.9) across both the cell lineand tumor samples were unified into a single vector prior to clustering.This unification resulted in 2351 non-redundant vectors from theoriginal 8939 pathway concepts. Samples were clustered using theresulting set of non-redundant concepts. The matrix of inferred pathwayactivities for both the 47 cell lines and 183 TCGA tumor samples wasclustered using complete linkage hierarchical agglomerative clusteringimplemented in the Eisen Cluster software package version 3.0⁴⁵Uncentered Pearson correlation was used as the metric for the pathwayconcepts and Euclidean distance was used for sample metric.

To quantify the degree to which cell lines clustered with tumor samplesof the same subtype, we compared two distributions of t-statisticsderived from Pearson correlations. Let C, be the set of cell lines ofsubtype s. Similarly, let T, be the set of TCGA tumor samples of subtypes. For example, C_(ba_), and 7′ b_(ay, a)l are the set of all basal celllines and basal tumor samples respectively. The first distribution wasmade up of t-statistics derived from the Pearson correlations betweenevery possible pair containing a cell line and tumor sample of the samesubtype; i.e. for all subtypes s, every pairwise correlationt-statistics was computed between a pair (a, b) such that a E C_(s) andb E T. The second distribution was made of correlation t-statisticsbetween cell lines of different subtypes; i.e. computed over pairs (a,b) such that a e C_(c) and b E C₅. and s s′. We performed aKohnogorov-Sminiov test to compare the distributions.

Example XXXIX: Molecular Subtypes of Tumors at Various Genetic MolecularLevels

The pioneering studies of whole genome gene expression analysisperformed on breast tumors have identified different subclasses mostnotably belonging to the estrogen receptor (ER) negative basal-like andthe ER positive luminal subgroups (Perou, C. M. et al., (2000),Molecular portraits of human breast tumours, 406: 747-752) withdifferences in clinical outcome (Sorlie, T. et al., (2001), Geneexpression patterns of breast carcinomas distinguish tumor subclasseswith clinical implications, 98: 10869-10874). The existence of severalmolecular subtypes has also been observed by DNA copy number analysis(2Russnes et al. (2007) supra), DNA methylation (Ronneberg et al. (2011)supra) and miRNA expression analyses (Enerly et al. (2011) supra).However, the questions are to what extent these new profiles, acquiredby molecular analyses at various new molecular levels, recapitulate theinitially discovered subclasses by mRNA expression, and what is thepotential of these new classifications to identify novel patientsubgroups of clinical importance? To address these questions we firstclustered the breast cancer patients of the MicMa dataset according toeach molecular level studied (FIG. 23) using an unbiased, unsupervisedmethod. The histograms of the clustering of patients by each molecularlevel separately and the survival KM plot for each patient subgroup areshown in FIG. 23. Interestingly, this clustering procedure lead to theidentification of 7 clusters of mRNA expression that correlated highlywith the clusters derived from Pam50 classification. It was consistentwith the Pam50, but split the Luminal A cluster between exp 1-4 mRNAclusters, and the basal and the ERBB2 among the last three (exp5-7)clusters. At the miRNA level three different clusters were obtained aspreviously described in (Enerly et al. (2011) supra); at methylationlevel three main clusters were seen as described and one much smaller,fourth cluster that was also observed but not further discussed inRonneberg et al. (2011, supra). At CNA level six different clustersappeared. Clearly, at every level the distinct patient clusters wereassociated with a particular pattern of survival (FIG. 23). Whether thesame patients formed the corresponding clusters at different molecularlevels was then evaluated. Indeed, there was to a great extent a goodconcordance between the clustering at different levels, most notablybetween DNA methylation and mRNA expression and DNA copy number (Table12). However, while some samples always cluster together at any level,others cluster in different groups according to each particularmolecular endpoint in study.

TABLE 12 mrna meth mir paradigm cna 1.38E−04 6.99E−03 9.09E−02 1.20E−05mrna 6.30E−05 4.12E−03 1.36E−09 meth 1.83E−01 1.26E−05 mir 2.57E−02

The consistent splitting of one subclass derived from one molecularlevel, by the clustering according to another may reveal importantbiological implications. For instance, as discussed in (3), while goodcorrelation between methylation and mRNA expression based classificationwas observed (p=2.29.10-6), still Luminal-A class (by mRNA expression)was split between two different methylation clusters. The same appliedto the basal-like tumors suggesting that despite the strong concordanceto the mRNA expression clusters additional information was provided bythe clustering according to DNA methylation. Luminal A samples withdifferent DNA methylation profiles differ in survival (3 Ronneberg, J.A. et al., (2011), Methylation profiling with a panel of cancer relatedgenes: association with estrogen receptor, TP53 mutation status andexpression subtypes in sporadic breast cancer, 5: 61-76). The increasingnumber of new datasets from both us and others will in the future revealwhether these clusters will converge to several most and many lessfrequent combinations.

Although reclassification at different molecular levels is worth offurther studies as it may point to new interesting biological pathwaysaffected on different levels, the information content in this horizontalreshuffling of samples from class to class may be limited. Looking atdifferentially expressed/altered genes within these clusters per pathwayis dependent on the a priori knowledge and choices of known interactionsand is unable to identify novel pathways. Further, these approachestreat genes and measurements in different datasets as independentvariables and do not take into consideration the position of a gene in apathway, or the number of its interactive partners (i.e. the pathway'stopology) and may be vulnerable to large fluctuations in the expressionof one or few genes in a gene set. It is commonly observed that aparticular pathway may be deregulated in many tumors in cancer, but thatthe particular gene and method of deregulation varies in differenttumors (Cancer Genome Atlas Research Network. Comprehensive genomiccharacterization defines human glioblastoma genes and core pathways.Nature 2008 October; 455(7216):1061-1068). We therefore next applied apathway based modeling methodology that models the interactions betweenthe different data type measurements on a single gene as well as knowninteractions between genes, in order to characterize each gene'sactivity level in a tumor in the context of a pathway and associatedclinical data. We used each gene's Integrated Pathway Levels (IPL) todirectly identify and classify the patients according to thesederegulated pathways (across molecular data types) and then investigatethe relationship of the new clusters with the previously describedclasses at various molecular levels.

Example XL: PARADIGM for Classification of Invasive Cancers withPrognostic Significance

In order to understand how genomic changes disturb distinct biologicalfunctions that can explain tumor phenotypes and make tumors vulnerableto targeted treatment, we need an understanding of perturbations at apathway level. PARADIGM identifies consistent active pathways in subsetsof patients that are indistinguishable if genes are studied at a singlelevel. The method uses techniques from probabilistic graphical models(PGM) to integrated functional genomics data onto a known pathwaystructure. It has previously been applied to analysis of copy number andmRNA expression data from the TCGA glioblastoma and ovarian datasets.PARADIGM analysis can also be used to connect genomic alterations atmultiple levels such as DNA methylation or copy number, mRNA and miRNAexpression and can thus integrate any number of omics layers of data ineach individual sample. Although DNA methylation and miRNA expressioncontribute to the observed here deregulated pathways and seem to havedistinct contribution to the prognosis and molecular profiles of breastcancer each in its own right in the MicMa cohort (FIG. 23) we did notfind improvement of the prognostic value of the PARADIGM clusters byadding these two molecular profile types. One explanation for this isthat the prognostic value of miRNA and DNA methylation analyses isrecapitulated by mRNA expression due to their high correlation. However,such conclusion requires further analysis regarding, for example,whether the choice of analysis platforms (limited Illumina 1505 CpGcancer panel for methylation) and our limited knowledge of true miRNAtargets may be the factors limiting our ability to comprehensivelymeasure and effectively model miRNA and DNA methylation information.

PARADIGM analyses based on mRNA expression and copy number alterationsof the MicMa cohort identified the existence of 5 different clusters(FIG. 24) and showed that combining mRNA expression and DNA copy numberleads to better discrimination of patients with respect to prognosisthan any of the molecular levels studied separately (FIG. 24 and FIG.23). The pathways whose perturbations most strongly contributed to thisclassification were those of Angiopoicntin receptor Tie2-mediatedsignaling and most notably the immune response (TCR) and interleukinsignaling, where nearly every gene or complex in the pathway deviatedfrom the normal (FIG. 25). Most prominently seen were IL4, IL6, IL12 andIL23 signaling. Other prominent pathways are Endothelins, FOXM1transcription, deregulated also in the ovarian and glioblastome TCGAdatasets and ERBB4, also previously found deregulated in breast andovarian cancers. Based on this analysis we have identified the followingpatients groups with significantly different prognosis, which can beroughly characterized as follows:

pdgm. I=high FOXM1, high immune signaling,

pdgm.2=high FOXM1, Low immune signaling, macrophage dominated,

pdgm.3=low FOXM1, low immune signaling,

pdgm.4=high ERBB4, low Angiopoietin signaling,

pdgm.5=high FOXM1, low macrophage signature.

The identification of the Paradigm clusters was validated in twopreviously published datasets, one by Chin et al 2007 (Chin, S. F. etal., (2007), Using array-comparative genomic hybridization to definemolecular portraits of primary breast cancers, 26: 1959-1970), whichcompared to the MicMa dataset was with higher frequency of ER- and highgrade tumors and even more interestingly in another set enriched for nonmalignant DCIS (Ductal carcinoma in situ) (12 Muggerud, A. A. et al.,(2010), Molecular diversity in ductal carcinoma in situ (DCIS) and earlyinvasive breast cancer, 4: 357-368) (FIG. 25). The heatmap for the pureDCIS tumors is shown in FIG. 25.

In the cluster with worst prognosis in MicMa, pdgm.2, ILA signaling isstrongly down-regulated in conjunction with STAT6, which has been shownin human breast cancer cells to prevent growth inhibition (16 Gooch, J.L., Christy, B., and Yee, D., (2002), STAT6 mediates interleukin-4growth inhibition in human breast cancer cells, 4: 324-331).Down-regulation of IL4 signaling has also promoted mast cell activationwhich can support greater tumor growth (17 de Visser, K. E., Eichten,A., and Coussens, L. M., (2006), Paradoxical roles of the immune systemduring cancer development, 6: 24-37). Conversely, in pdgm.5, macrophageactivation is decreased and natural killer cell activity is increaseddue to IL23 signaling. A cancer dependent polarization of the immuneresponse towards Th-2 and B cells recruitment on one side and Th-1proliferation on the other, has been discussed (1 Ursini-Siegel, J. etal., (2010), Receptor tyrosine kinase signaling favors a protumorigenicstate in breast cancer cells by inhibiting the adaptive immune response,70: 7776-7787). It has been hypothesized that under certain conditionsTh1/CTL immune response may prevent the transition of hyperplasia toadenoma in mice, while Th2 response may by conferring a chronicinflammatory state to promote the transition to carcinoma. I L4 is aTh-2 derived cytokine that stimulates B cells differentiation andchronic inflammation in cancer cells. Further Th-2 cells secrete IL10that mediates immunosuppression in these cancers. This immunosuppressionwas shown to occur predominantly in basal and ERBB2 cancers. In supportto this, it has been shown recently that “antitumor acquired immuneprograms can be usurped in pro-tumor microenvironments and insteadpromote malignancy by engaging cellular components of the innate immunesystem functionally involved in regulating epithelial cell behavior”(DeNardo, D. G. et al., (2009), CD4(+) T cells regulate pulmonarymetastasis of mammary carcinomas by enhancing protumor properties ofmacrophages, 16: 91-102).

There was a considerable concordance between this immunoclassification,proposed here and the well established classification by mRNA expression(luminal A,B, basal, ERBB2, normal like) (FIG. 24. Samples belonging tothe basal and ERBB2 clusters were of predominantly prgm1 (worseprognosis), Luminal A—prgm 3 (best prognosis). The Paradigm clusteringoffers however a rather significant distinction between luminal A(prgm3) and luminal B (prgm4) clusters, as well as the identification ofa subset of basal tumors with very bad prognosis (prgm2).

Example XLI: Identified Pathways Whose Perturbation SpecificallyInfluences the PARADIGM Clustering

FOXM1 Transcription.

FOXM1 is a key regulator of cell cycle progression and its endogenousFOXM1 expression oscillates according to the phases of the cell cycle.FOXM1 confirmed as a human proto-oncogene is found upregulated in themajority of solid human cancers including liver, breast, lung, prostate,cervix of uterus, colon, pancreas, brain as well as basal cellcarcinoma, the most common human cancer. FOXM1 is thought to promoteoncogenesis through its multiple roles in cell cycle andchromosomal/genomic maintenance (Wonsey, D. R. and Follettie, M. T.,(2005), Loss of the forkhead transcription factor FOXM1 causescentrosome amplification and mitotic catastrophe, 65: 5181-5189).Aberrant upregulation of FOXM1 in primary human skin keratinocytes candirectly induce genomic instability in the form of loss ofheterozygosity (LOH) and copy number aberrations (Teh M, Gemenetzidis E,Chaplin T, Young B D, Philpott M P. Upregulation of FOXM1 inducesgenomic instability in human epidermal keratinocytes. Mol. Cancer 2010;9:45). A recent report showed that aberrant upregulation of FOXM1 inadult human epithelial stem cells induces a pre-cancer phenotype in a3D-organotypic tissue regeneration system—a condition similar to humanhyperplasia (Gemenetzidis, E. et al., (2010), Induction of humanepithelial stem/progenitor expansion by FOXM1, 70: 9515-952). Theauthors showed that excessive expression of FOXM1 exploits the inherentself-renewal proliferation potential of stem cells by interfering withthe differentiation pathway, thereby expanding the progenitor cellcompartment. It was therefore hypothesized that FOXM1 induces cancerinitiation through stem/progenitor cell expansion. We see clearly twogroups of breast cancer patients with high and low activity of thispathway, broken mainly according to interleukin signaling activity. FIG.26 illustrates the opposite activation modus of this pathway (red asactivated vs blue inactivated) for cluster pdgm 3 (best survival) asopposed to the rest of the clusters with worse survival and themolecular levels that contribute to it (mRNA, CNA, miRNA or DNAmethylation according to the shape of the figures). One can notice thatdown regulation of MMP2 in pdgm3 is due to DNA methylation, while in therest of the tumors—due to DNA deletion. Of the miRNAs, has-let?-b wasupregulated in pgm3 and downregulated in the rest, complementary to itstarget, the AURKB. Both DNA amplification and mRNA expression were seenas causes of deregulation of expression.

Angiopoietin Receptor Tie2-Mediated Signaling.

The Ang family plays an important role in angiogenesis during thedevelopment and growth of human cancers. Ang2's role in angiogenesisgenerally is considered as an antagonist for Ang I, inhibiting Ang1-promoted Tie2 signaling, which is critical for blood vessel maturationand stabilization (23). Ang2 modulates angiogenesis in a cooperativemanner with another important angiogenic factor, vascular endothelialgrowth factor A (VEGFA) (Hashizume, H. et al., (2010), Complementaryactions of inhibitors of angiopoietin-2 and VEGF on tumor angiogenesisand growth, 70: 2213-2223). New data suggests more complicated roles forAng2 in angiogenesis in invasive phenotypes of cancer cells duringprogression of human cancers. Certain angiopoietin (Ang) family memberscan activate Tie1, for example, Ang I induces Tie1 phosphorylation inendothelial cells (2 Yuan, H. T. et al., (2007), Activation of theorphan endothelial receptor Tie1 modifies Tie2-mediated intracellularsignaling and cell survival, 21: 3171-3183). Tie1 phosphorylation is,however, Tie2 dependent because Ang1 fails to induce Tie1phosphorylation when Tie2 is down-regulated in endothelial cells andTie1 phosphorylation is induced in the absence of Ang 1 by either aconstitutively active form of Tie2 or a Tie2 agonistic antibody (25 Yuanet al. (2007) supra). Ang 1-mediated AKT and 42/44MAPK phosphorylationis predominantly Tie2 mediated, and Tie1 down-regulates this pathway.Thus the main role for Tie1 is to modulate blood vessel morphogenesisdue to its ability to down-regulate Tie2-driven signaling andendothelial survival. Both Tie2 mediated signaling as well as VEGFR1 and2 mediated signaling and specific signals were observed in this dataset.

ERBB4

ERBB4 contributes to proliferation and cell movements in mammarymorphogenesis and the directional cell movements of Erbb4-expressingmammary primordial epithelia while promoting mammary cell fate.Candidate effectors of Nrg3/Erbb4 signaling have been identified andshown here to interacts with other signalling pathways relevant to earlymammary gland development and cancer. One of the primary functions ofErbB4 in vivo is in the maturation of mammary glands during pregnancyand lactation induction. Pregnancy and extended lactation durations havebeen correlated with reduced risk of breast cancer, and the role ofErbB4 in tumor suppression may therefore be linked with its role inlactation. Most reports are consistent with a role for ErbB4 inreversing growth stimuli triggered by other ErbB family members duringpuberty, however significant association of survival to ERBB4 expressionhas not been confirmed (2 Sundvall, M. et al., (2008), Role of ErbB4 inbreast cancer, 13: 259-268).

Example XLII: PARADIGM for Classification in Ductal Carcinoma In Situ(DCIS)

Given the involvement of immune response in premalignant hyperplasticglands in mouse models (18 Ursini-Siegel, J. et al., (2010), Receptortyrosine kinase signaling favors a protumorigenic state in breast cancercells by inhibiting the adaptive immune response, 70: 7776-7787), weanalyzed a previously published dataset comprising of DCIS cases to findwhether the observed strong immune response and interleukin signaling ininvasive tumors is present in pre-malignant stages as well. Ductalcarcinoma in situ (DCIS) is a non-invasive form of breast cancer wheresome lesions are believed to rapidly transit to invasive ductalcarcinomas (IDCs), while others remain unchanged. We have previouslystudied gene expression patterns of 31 pure DCIS, 36 pure invasivecancers and 42 cases of mixed diagnosis (invasive cancer with an in situcomponent) (1Muggerud et al. (2010) supra) and observed heterogeneity inthe transcriptomes among DCIS of high histological grade, identifying adistinct subgroup of DCIS with gene expression characteristics moresimilar to advanced tumors. The heatmap, of the PARADIGM results forthis entire cohort (including IDC and ILC) in FIG. 25 and for the pureDCIS samples, in FIG. 25. None of the pure DCIS tumors were of prgm2type, characterized by signaling typical for high macrophage activity(FIG. 25). In agreement, experimental studies have demonstrated thatmacrophages in primary mammary adenocarcinomas regulate late-stagecarcinogenesis thanks to their proangiogenic properties (Lin, E. Y. andPollard, J. W., (2007), Tumor-associated macrophages press theangiogenic switch in breast cancer, 67: 5064-5066; Lin, E. Y. et al.,(2007), Vascular endothelial growth factor restores delayed tumorprogression in tumors depleted of macrophages, 1: 288-302), as well asfoster pulmonary metastasis by providing epidermal growth factor (EGF)to malignant mammary epithelial cells. Again among the top deregulatedpathways identified by the PARDIGM analysis in DCIS were those involvingIL2, 4, 6, 12, 23, and 23 signaling.

In both datasets (DCIS, MicMa) TCR signaling in naive CD8+ T cells wason top of the list alongside with a large number of chemokines that areknown to recruit CD8+ T cells. One is IL-12, produced by the antigenpresenting cells that was shown to stimulate IFN-gamma production fromNK and T cells. IFN-gamma pathway was one of the deregulated pathways,higher up on the list in DCIS. IFNgamma is produced from the Th1 cellsand the NK cells and was shown to initiate an antitumor immune response.Phase I clinical trials have shown that the clinical effect oftrastuzumab (herceptin) is potentiated by the co-administration of IL-12to patients with HER2-overexpressing tumors, and this effect is mediatedby the stimulation of IFNgamma production in the NK cells (29). In DCIS,other most strong contributor (Table 8) was 84 NOX4. NOX4, anoxygen-sensing NAPHD oxidase, and a phagocyte-type A oxidase, is similarto that responsible for the production of large amounts of reactiveoxygen species (ROS) in neutrophil granulocytes, primary immuneresponse. Also FN1 (fibronectin) and PDGFRB, the platelet-derived growthfactor receptor, appeared repeatedly together specifically in the DCIStogether with COL1A2, IL12/1L12R/TYK2/JAK2/SPHK2, ESR1 and ICRT14.

These genes/pathways seem to be all contributing to functions in theextracellular matrix, the cell-cell interaction, and fibrosis andkeratinization. For instance, FN1 Fibronectin-1 belongs to a family ofhigh molecular weight glycoproteins that are present on cell surfaces,in extracellular fluids, connective tissues, and basement membranes.Fibronectins interact with other extracellular matrix proteins andcellular ligands, such as collagen, fibrin, and integrins. Fibronectinsare involved in adhesive and migratory processes of cells. PDGFR, theplatelet-derived growth factor receptor, together with the Epidermalgrowth factor (EGF) signals through EGF and PDGF receptors, which areimportant receptor tyrosine kinases (RTKs). Importantly, PDGFR foundhere to be overexpressed in certain DCIS is a target of Sunitinib (30Fratto, M. E. et al., (2010), New perspectives: role of sunitinib inbreast cancer, 161: 475-482) and a secondary target of Imatinib mesylate(Gleevec) (Weigel, M. T. et al., (2010), In vitro effects of imatinibmesylate on radiosensitivity and chemosensitivity of breast cancercells, 10: 412). Contrary to the immunostimulatory role of trastuzumab(herceptin) described above to mediated by increased INFgammaproduction, imatinib was shown to inhibit interferon-gamma production byTCR-activated CD4(+) T cells. These observations are of interest for ourargument to the degree that they illuminate the interaction betweengrowth factor receptors presented on the surface of DCIS and malignantcells and immune constitution. It was shown that stimulatoryautoantibodies to PDGFR appeared to trigger an intracellular loop thatinvolves Ras, ERK1/ERK2, and reactive oxygen species (ROS) that leads toincreased type I collagen expression. This is in line with COL1A2expression also observed as deregulated in DCIS in our study.

Example XLIII: Materials and Methods

The analysis was applied to data collected from ca 110 breast carcinomaswith mRNA expression analyzed by Agilent whole human genome 4×44K onecolor oligo array. The copy number alterations (CNA) was analyzed usingthe Illumina Human-1 109K BeadChip. This SNP array is gene centric andcontains markers covering the entire genome with an average physicaldistance of 30 kb and represents 15,969 unique genes (May 2004 assembly,hg17, NCBI Build 35). Each sample was subjected to whole genomeamplification. Genotype reports and logR values were extracted withreference to dbSNP's (build 125) forward allele orientation usingBeadStudio (v. 2.0, Illumina), and logR values were adjusted for CNAs.

miRNA profiling from total RNA was performed using Agilent Technologies“Human miRNA Microarray Kit (V2)” according to manufacturer's protocol.Scanning on Agilent Scanner G2565A and Feature Extraction (FE) v9.5 wasused to extract signals. Experiments were performed using duplicatehybridizations (99 samples) on different arrays and time points. Twosamples were profiled only once. miRNA signal intensities for replicateprobes were averaged across the platform, log 2 transformed andnormalized to the 75 percentile. miRNA expression status was scored aspresent or absent for each gene in each sample by default settings in FEv9.5.

DNA methylation, One microgram of DNA was bisulphite treated using theEpiTect 96 Bisulfite Kit (Qiagen GmbH, Germany). 500 ng of bisulphitetreated DNA was analyzed using the GoldenGate Methylation Cancer Panel I(Illumina Inc, CA, USA) that simultaneously analyses 1505 CpG sites in807 cancer related genes. At least 2 CpG sites were analyzed per genewere one CpG site is in the promoter region and one CpG site is in the1st exon Bead studio software was used for the initial processing of themethylation data according to the manufacturer's protocol. The detectionp-value for each CpG site was used to validate sample performance andthe dataset was filtered based on the detection p-value were CpG siteswith a detection p-value>0.05 was omitted from further analysis.

Data pre-processing and Paradigm parameters. Copy number was segmentedusing CBS, then mapped to gene-level measurements by taking the medianof all segments that span a RefSeq gene's coordinates in hg18. For mRNAexpression, measurements were first probe-normalized by subtracting themedian expression value for each probe. The manufacturer's genomiclocation for each probe was converted from hg17 to hg18 using UCSCsliftOver tool. Per-gene measurements were then obtained by taking themedian value of all probes overlapping a RefSeq gene. Methylation probeswere matched to genes using manufacturers description. Paradigm was runas previously (10), by quantile transforming each data set separately,but data was discretized into bins of equal size, rather than at the 5%and 95% quantiles. Pathway files were from the PID (36) as previouslyparsed. FIG. 26 shows summaries of discretized input data, and not rin,values, by counting the fraction of observations in either an up or downbin in each datatype, and then labeling each node with the bin with thehighest fraction of observations in any datatype.

HOPACH Unsupervised Clustering. Clusters were derived using the HOPACH Rimplementation version 2.10 (37) running on R version 2.12. Thecorrelation distance metric was used with all data types, except forParadigm IPLs, which used cosangle duc to the non-normal distributionand prevalence of zero values. For any cluster of samples that containedfewer than 5 samples, each sample was mapped to the same cluster as themost similar sample in a larger cluster. Paradigm clusters in the MicMadataset were mapped to other datatypes by determining each cluster'smediod (using the median function) in the MicMa dataset, then assigningeach sample in another dataset to whichever cluster mediod was closestby cosangle distance.

Kaplain-Meier, Cluster enrichments. Kaplan-Meier statistics, plots, andcluster enrichments were determined using R version 2.12. Cox p-valueswere determined using the Wald test from the coxph( ) proportionalhazards model, and log-rank p-values from a chi-square test from thesurvdiff( ) function. Overall enrichment of a gene's or pathway member'svalues for a clustering were determined by ANOVA, and enrichment of agene for a particular cluster label were determined by a T-test of agene's values in a particular cluster vs. the gene's values in all otherclusters. FDR was determined using the Benjamini &Hochberg method ofp.adjust.

Example XLIV: Data Sets and Pathway Interactions

Both copy number and expression data were incorporated into PARADIGMinference. Since a set of eight normal tissue controls was available foranalysis in the expression data, each patient's gene-value wasnormalized by subtracting the gene's median level observed in the normalfallopian control. Copy number data was normalized to reflect thedifference in copy number between a gene's level detected in tumorversus a blood normal. For input to PARADIGM, expression data was takenfrom the same integrated dataset used for subtype analysis and the copynumber was taken from the segmented calls of MSKCC Agilent 1M copynumber data.

A collection of pathways was obtained from NCI-PID containing 131pathways, 11,563 interactions, and 7,204 entities. An entity ismolecule, complex, small molecule, or abstract concept represented as“nodes” in PARADIGM's graphical model. The abstract concepts correspondto general cellular processes (such as “apoptosis” or “absorption oflight,”) and families of genes that share functional activity such asthe RAS family of signal transducers. We collected interactionsincluding protein-protein interactions, transcriptional regulatoryinteractions, protein modifications such as phosphorylation andubiquitinylation interactions.

Example XLV: Inference of Integrated Molecular Activities in PathwayContext

We used PARADIGM, which assigns an integrated pathway activity (IPA)reflecting the copy number, gene expression, and pathway context of eachentity.

The significance of IPAs was assessed using permutations of gene- andpatient-specific cross-sections of data. Data for 1000 “null” patientswas created by randomly selecting a gene-expression and copy number pairof values for each gene in the genome. To assess the significance of thePARADIGM IPAs, we constructed a null distribution by assigning randomgenes to pathways while preserving the pathway structure.

Example XLVI: Identification of FOXM1 Pathway

While all of the genes in the FOXM1 network were used to assess thestatistical significance during the random simulations, in order toallow visualization of the FOXM1 pathway, entities directly connected toFOXM1 with significantly altered IPAs according to FIG. 29 were chosenfor inclusion in FIG. 27. Among these, genes with roles in DNA repairand cell cycle control found to have literature support for interactionswith FOXM1 were displayed. BRCC complex members, not found in theoriginal NCI-PID pathway, were included in the plot along with BRCA2,which is a target of FOXM1 according to NCI-PID. Upstream DNA repairtargets were identified by finding upstream regulators of CHEK2 in otherNCI pathways (for example, an indirect link from ATM was found in thePLK3 signaling pathway).

Example XLVII: Clustering

The use of inferred activities, which represent a change in probabilityof activity and not activity directly, it enables entities of varioustypes to be clustered together into one heatmap. To globally visualizethe results of PARADIGM inference, Eisen Cluster 3.0 was used to performfeature filtering and clustering. A standard deviation filtering of 0.1resulted in 1598 out of 7204 pathway entities remaining, and averagelinkage, uncentered correlation hierarchical cluster was performed onboth the entities and samples.

Example XLVIII Isolation of Genomic DNA

Blood samples (2-3 ml) are collected from patients and stored inEDTA-containing tubes at −80° C. until use. Genomic DNA is extractedfrom the blood samples using a DNA isolation kit according to themanufacturer's instruction (PUREGENE, Gentra Systems, MinneapolisMinn.). DNA purity is measured as the ratio of the absorbance at 260 and280 nm (1 cm lightpath; A₂₆₀/A₂₈₀) measured with a Beckmanspectrophotometer.

Example XLIX: Identification of SNPs

A region of a gene from a patient's DNA sample is amplified by PCR usingthe primers specifically designed for the region. The PCR products aresequenced using methods well known to those of skill in the art, asdisclosed above. SNPs identified in the sequence traces are verifiedusing Phred/Phrap/Consed software and compared with known SNPs depositedin the NCBI SNP databank.

Example L: Statistical Analysis

Values are expressed as mean±SD. x² analysis (Web Chi Square Calculator,Georgetown Linguistics, Georgetown University, Washington D.C.) is usedto assess differences between genotype frequencies in normal subjectsand patients with a disorder. One-way ANOVA with post-hoc analysis isperformed as indicated to compare hemodynamics between different patientgroups.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein. Therefore, itis to be understood that the invention can be practiced other than asspecifically described herein. The above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A pathway analysis computing system comprising: acomputer readable, non-transitory memory storing software instructionsand a probabilistic graphical model (PGM) of a gene expression pathwaynetwork, the PGM including: directed graphs that include pathwayinteractions and that include hidden cell states and observed cellstates of biological cellular entities for a set of genes, wherein thebiological cellular entities include gene copy number, mRNA expression,protein level, and protein activity for each of the set of genes, andwherein measurements are available for the observed cell states and notavailable for the hidden cell states; wherein the directed graphs form adata structure including nodes representing the biological cellularentities and edges representing the pathway interactions among the nodesof the biological cellular entities associated with the pathwayinteractions; and wherein variables of the directed graphs representdifferential states of the hidden cell states and the observed cellstates relative to corresponding normal levels of the biologicalcellular entities, and where factors of edges of the directed graphs areassigned to the edges representing the pathway interactions between thebiological cellular entities, and wherein the pathway interactions arebiochemical interactions; and a processor coupled with the memory andconfigured, upon execution of the software instructions, to outputintegrated pathway activities for pathways associated with a patient by:initializing the PGM by assigning, in the memory, values measured from atissue sample of the patient to the observed cell states of observednodes in the directed graphs; simulating the gene expression pathwaynetwork, using the initialized PGM, to estimate the hidden cell statesof hidden nodes of the directed graphs for the tissue sample based onthe assigned observed cell states in the directed graphs and accordingto the factors of edges of the directed graphs that connect the hiddennodes and the observed nodes, wherein the simulating includes:determining a probability of a given hidden cell state for each hiddennode of the hidden nodes based on (1) probabilities of cell states ofparent nodes of the hidden node and (2) the factors of edges from theparent nodes, and iteratively updating the hidden cell states based onthe probabilities of the cell states and the factors of edges; andoutputting, from the memory, the estimated hidden cell states of thebiological cellular entities for the tissue sample as integrated pathwayactivities of the tissue sample, wherein the estimated hidden cellstates include a protein activity.
 2. The system of claim 1, wherein theprobabilistic graphical model comprises at least two directed graphs. 3.The system of claim 1, wherein the directed graphs comprise a factorgraphs.
 4. The system of claim 1, wherein the directed graphs comprise aBayesian network.
 5. The system of claim 1, wherein the pathways areformatted according to BioPAX formatting rules.
 6. The system of claim5, wherein the pathways are formatted according to BioPAX level
 2. 7.The system of claim 1, wherein the biological cellular entities includeat least four different biological cellular entities.
 8. The system ofclaim 1, wherein the integrated pathway activities include at leastthree states for the biological cellular entities.
 9. The system ofclaim 8, wherein the at least three states include: an active state, aninactive state, and a nominal state.
 10. The system of claim 1, whereinthe estimated hidden cell states are estimated by belief propagation.11. The system of claim 1, wherein the estimated hidden cell states areestimated by iterating between inferring probabilities of the hiddencell states and updating the hidden cell states to maximize likelihoodgiven probabilities of the hidden cell states.
 12. The system of claim11, wherein the estimated hidden cell states are estimated via animplementation of an expectation-maximization algorithm.
 13. The systemof claim 1, wherein the factors between the biological cellular entitiesinclude non-negative functions.
 14. The system of claim 13, wherein thenon-negative functions constrain values of the cell states.
 15. Thesystem of claim 1, wherein the factors define probabilities associatedwith states of the biological cellular entities.
 16. The system of claim1, wherein the gene expression pathway network includes at least one of:a regulatory pathway network and a signaling pathway network.
 17. Thesystem of claim 1, wherein the values obtained from the tissue sample ofthe patient and assigned to the observed cell states include at leastone of the following: mutation, a differential genetic sequence object,a gene copy number, a transcription level, a translation level, aprotein activity, and a protein interaction.
 18. The system of claim 1,wherein the tissue sample is at least one of the following: a diseasedtissue, a normal tissue, an aging tissue, a recovering tissue, an animaltissue, an experimentally treated tissue, a cancer tissue, and a patientfluid.
 19. The system of claim 1, wherein the PGM is generated usingobserved cell states of a cohort of patients that have a same conditionas the patient.
 20. A method comprising: measuring values from a tissuesample of a patient, wherein the measured values correspond to observedcell states and not to hidden cell states; storing, by a computer in adatabase, a probabilistic graphical model (PGM) of a gene expressionpathway network, the PGM including: directed graphs that include pathwayinteractions and that include hidden cell states and observed cellstates of biological cellular entities for a set of genes, wherein thebiological cellular entities include gene copy number, mRNA expression,protein level, and protein activity for each of the set of genes, andwherein measurements are available for the observed cell states and notavailable for the hidden cell states; wherein the directed graphs form adata structure including nodes representing the biological cellularentities and edges representing the pathway interactions among the nodesof the biological cellular entities associated with the pathwayinteractions; and wherein variables of the directed graphs representdifferential states of the hidden cell states and the observed cellstates relative to corresponding normal levels of the biologicalcellular entities, and where factors of edges of the directed graphs areassigned to the edges representing the pathway interactions between thebiological cellular entities, and wherein the pathway interactions arebiochemical interactions; initializing, by the computer, the PGM byassigning values measured from the tissue sample of the patient to theobserved cell states of observed nodes in the directed graphs;simulating, by the computer, the gene expression pathway network, usingthe initialized PGM, to estimate the hidden cell states of hidden nodesof the directed graphs for the tissue sample based on the assignedobserved cell states in the directed graphs and according to the factorsof edges of the directed graphs that connect the hidden nodes and theobserved nodes, wherein the simulating includes: determining aprobability of a given hidden cell state for each hidden node of thehidden nodes based on (1) probabilities of cell states of parent nodesof the hidden node and (2) the factors of edges from the parent nodes,and iteratively updating the hidden cell states based on theprobabilities of the cell states and the factors of edges; andoutputting, by the computer, the estimated hidden cell states of thebiological cellular entities for the tissue sample as integrated pathwayactivities of the tissue sample, wherein the estimated hidden cellstates include a protein activity.
 21. The method of claim 20, furthercomprising: determining integrated pathway activities by comparing theprobabilities of the estimated hidden cell states determined using theobserved cell states and the factors to prior probabilities of hiddenstates iteratively determined without the observed cell states, whereinoutputting the estimated hidden cell states is accomplished byoutputting the integrated pathway activities.
 22. The system of claim 1,wherein the processor is further configured, upon execution of thesoftware instructions, to perform: determining integrated pathwayactivities by comparing the probabilities of the estimated hidden cellstates determined using the observed cell states and the factors toprior probabilities of hidden states iteratively determined without theobserved cell states, wherein outputting the estimated hidden cellstates is accomplished by outputting the integrated pathway activities.23. The system of claim 1, wherein the probabilistic graphical modelincludes at least 55 nodes.